Biphenyl compounds useful as muscarinic receptor antagonists

ABSTRACT

This invention provides compounds of formula I:  
                 
 
wherein a, b, c, d, m, n, p, s, t, W, Ar 1 , R 1 , R 2 , R 3 , R 4 , R 6 , R 7 , and R 8  are as defined in the specification. The compounds of formula I are muscarinic receptor antagonists. The invention also provides pharmaceutical compositions containing such compounds, processes and intermediates for preparing such compounds and methods of using such compounds to treat pulmonary disorders.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/552,443, filed on Mar. 11, 2004; the entire disclosure of which isincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to novel biphenyl compounds havingmuscarinic receptor antagonist or anticholinergic activity. Thisinvention also relates to pharmaceutical compositions comprising suchbiphenyl compounds, processes and intermediates for preparing suchbiphenyl compounds and methods of using such biphenyl compounds to treatpulmonary disorders.

2. State of the Art

Pulmonary or respiratory disorders, such as chronic obstructivepulmonary disease (COPD) and asthma, afflict many millions of peopleworldwide and such disorders are a leading cause of morbidity andmortality.

Muscarinic receptor antagonists are known to provide bronchoprotectiveeffects and therefore, such compounds are useful for treatingrespiratory disorders, such as COPD and asthma. When used to treat suchdisorders, muscarinic receptor antagonists are typically administered byinhalation. However, even when administered by inhalation, a significantamount of the muscarinic receptor antagonist is often absorbed into thesystemic circulation resulting in systemic side effects, such as drymouth, mydriasis and cardiovascular side effects.

Additionally, many inhaled muscarinic receptor antagonists have arelatively short duration of action requiring that they be administeredseveral times per day. Such a multiple-daily dosing regime is not onlyinconvenient but also creates a significant risk of inadequate treatmentdue to patient non-compliance with the required frequent dosingschedule.

Accordingly, a need exists for new muscarinic receptor antagonists. Inparticular, a need exists for new muscarinic receptor antagonists thathaving high potency and reduced systemic side effects when administeredby inhalation. Additionally, a need exists for inhaled muscarinicreceptor antagonists having a long duration of action thereby allowingfor once-daily or even once-weekly dosing. Such compounds are expectedto be particularly effective for treating pulmonary disorders, such asCOPD and asthma, while reducing or eliminating side effects, such asdry-mouth and constipation.

SUMMARY OF THE INVENTION

The present invention provides novel biphenyl compounds havingmuscarinic receptor antagonist or anticholinergic activity. Among otherproperties, compounds of this invention have been found to possess highpotency and reduced systemic side effects when administered byinhalation and to have a long duration of action.

Accordingly, in one of its composition aspects, this invention providesa compound of formula I:

wherein:

a is 0 or an integer of from 1 to 5;

each R¹ is independently selected from (1-4C)alkyl, (2-4C)alkenyl,(2-4C)alkynyl, (3-6C)cycloalkyl, cyano, halo, —OR^(1a), —C(O)OR^(1b),—SR^(1c), —S(O)R^(1d), —S(O)₂R^(1e), —NR^(1f)R^(1g),—NR^(1h)S(O)₂R^(1i), and —NR^(1j)C(O)R^(1k); where each of R^(1a),R^(1b), R^(1c), R^(1d), R^(1e), R^(1f), R^(1g), R^(1h), R^(1i), R^(1j),and R^(1k) is independently hydrogen, (1-4C)alkyl or phenyl(1-4C)alkyl;

b is 0 or an integer of from 1 to 4;

each R² is independently selected from (1-4C)alkyl, (2-4C)alkenyl,(2-4C)alkynyl, (3-6C)cycloalkyl, cyano, halo, —OR^(2a),—C(O)OR^(2b),SR^(2c)S(O)R^(2d)S(O)₂R^(2e), —NR^(2f)R^(2g),—NR^(2h)S(O)₂R^(2i), and —NR^(2j)C(O)R^(2k); where each of R^(2a),R^(2b), R^(2c), R^(2d), R^(2e), R^(2f), R^(2g), R^(2h), R^(2i), R^(2j),and R^(2k) is independently hydrogen, (1-4C)alkyl or phenyl(1-4C)alkyl;

W represents O or NW^(a), where W^(a) is hydrogen or (1-4C)alkyl;

c is 0 or an integer from 1 to 5;

each R³ independently represents (1-4C)alkyl or two R³ groups are joinedto form (1-3C)alkylene, (2-3C)alkenylene or oxiran-2,3-diyl;

m is 0 or 1;

R⁴ is selected from hydrogen, (1-4C)alkyl, and (3-4C)cycloalkyl;

s is 0, 1 or 2;

Ar¹ represents a phenylene group or a (3-5C)heteroarylene groupcontaining 1 or 2 heteroatoms independently selected from oxygen,nitrogen or sulfur; wherein the phenylene or heteroarylene group issubstituted with (R⁵)_(q) where q is 0 or an integer from 1 to 4 andeach R⁵ is independently selected from halo, hydroxy, (1-4C)alkyl or(1-4C)alkoxy;

t is 0, 1 or 2;

n is 0 or an integer from 1 to 3;

d is 0 or an integer from 1 to 4;

each R⁶ independently represents fluoro or (1-4C)alkyl;

p is 0 or 1; and

R⁷ and R⁸ are independently hydrogen or (1-4C)alkyl;

wherein each alkyl and alkoxy group in R¹, R^(1a-1k), R², R^(2a-2k), R³,R⁵, R⁶, R⁷, and R⁸ is optionally substituted with 1 to 5 fluorosubstituents;

or a pharmaceutically acceptable salt or solvate or stereoisomerthereof.

In another of its composition aspects, this invention is directed to apharmaceutical composition comprising a pharmaceutically acceptablecarrier and a therapeutically effective amount of a compound of formulaI or a pharmaceutically acceptable salt or solvate or stereoisomerthereof. Such pharmaceutical compositions may optionally contain othertherapeutic agents. Accordingly, in one embodiment, this invention isdirected to such a pharmaceutical composition wherein the compositionfurther comprises a therapeutically effective amount of a steroidalanti-inflammatory agent, such as a corticosteroid; a β₂ adrenergicreceptor agonist; a phosphodiesterase-4 inhibitor; or a combinationthereof.

Compounds of this invention possess muscarinic receptor antagonistactivity. Accordingly, compounds of formula I are expected to be usefulfor treating pulmonary disorders, such as chronic obstructive pulmonarydisease and asthma.

Accordingly, in one of its method aspects, this invention is directed toa method for treating a pulmonary disorder, the method comprisingadministering to a patient a therapeutically effective amount of acompound of formula I or a pharmaceutically acceptable salt or solvateor stereoisomer thereof.

Additionally, in another of its method aspects, this invention isdirected to a method of producing bronchodilation in a patient, themethod comprising administering to a patient a bronchodilation-producingamount of a compound of formula I or a pharmaceutically acceptable saltor solvate or stereoisomer thereof.

This invention is also directed to a method of treating chronicobstructive pulmonary disease or asthma, the method comprisingadministering to a patient a therapeutically effective amount of acompound of formula I or a pharmaceutically acceptable salt or solvateor stereoisomer thereof.

In another one of its method aspects, this invention is directed to amethod for antagonizing a muscarinic receptor in a mammal comprisingadministering to the mammal, a therapeutically effective amount of thecompound of formula I.

Since compounds of this invention possess muscarinic receptor antagonistactivity, such compounds are also useful as research tools. Accordingly,in yet another of its method aspects, this invention is directed to amethod for using a compound of formula I or a pharmaceuticallyacceptable salt or solvate or stereoisomer thereof as a research toolfor studying a biological system or sample, or for discovering newchemical compounds having muscarinic receptor antagonist activity.

This invention is also directed to processes and novel intermediatesuseful for preparing compounds of formula I or a pharmaceuticallyacceptable salt or solvate or stereoisomer thereof. Accordingly, inanother of its method aspects, this invention is directed to a processof preparing a compound of formula I, the process comprising:

-   -   (a) reacting a compound of formula II with a compound of formula        III; or    -   (b) coupling a compound of formula IV with a compound of formula        V; or    -   (c) reacting a compound of formula VI with a compound of formula        VII; or    -   (d) reacting a compound of formula II with a compound of formula        VIII in the presence of a reducing agent; or    -   (e) reacting a compound of formula IX with a compound of formula        VII in the presence of a reducing agent; or    -   (f) reacting a compound of formula XVIII with a compound of        formula XIX; and then removing any protecting groups, if        necessary, to provide a compound of formula I; wherein compounds        of formula I-IX, XVIII and XIX, are as defined herein.

In one embodiment, the above process further comprises the step offorming a pharmaceutically acceptable salt of a compound of formula I.In other embodiments, this invention is directed to the other processesdescribed herein; and to the product prepared by any of the processesdescribed herein.

This invention is also directed to a compound of formula I or apharmaceutically acceptable salt or solvate or stereoisomer thereof, foruse in therapy or as a medicament.

Additionally, this invention is directed to the use of a compound offormula I or a pharmaceutically acceptable salt or solvate orstereoisomer thereof, for the manufacture of a medicament; especiallyfor the manufacture of a medicament for the treatment of a pulmonarydisorder or for antagonizing a muscarinic receptor in a mammal.

DETAILED DESCRIPTION OF THE INVENTION

In one of its composition aspects, this invention is directed to novelbiphenyl compounds of formula I or pharmaceutically acceptable salts orsolvates or stereoisomers thereof. These compounds may contain one ormore chiral centers and therefore, this invention is directed to racemicmixtures; pure stereoisomers (i.e., enantiomers or diastereomers);stereoisomer-enriched mixtures and the like unless otherwise indicated.When a particular stereoisomer is shown or named herein, it will beunderstood by those skilled in the art that minor amounts of otherstereoisomers may be present in the compositions of this inventionunless otherwise indicated, provided that the desired utility of thecomposition as a whole is not eliminated by the presence of such otherisomers.

The compounds of formula I also contain several basic groups (e.g.,amino groups) and therefore, the compounds of formula I can exist as thefree base or in various salt forms. All such salt forms are includedwithin the scope of this invention. Furthermore, solvates of compoundsof formula I or salts thereof are included within the scope of thisinvention.

Additionally, where applicable, all cis-trans or E/Z isomers (geometricisomers), tautomeric forms and topoisomeric forms of the compounds offormula I are included within the scope of this invention unlessotherwise specified.

The compounds of formula I, as well as those compounds used in itssynthesis, may also include isotopically-labeled compounds, i.e., whereone or more atoms have been enriched with atoms having an atomic massdifferent from the atomic mass predominately found in nature. Examplesof isotopes that may be incorporated into the compounds of Formula (I)include, but are not limited to, ²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O and ¹⁷O.

The nomenclature used herein to name the compounds of this invention isillustrated in the Examples herein. This nomenclature has been derivedusing the commercially-available AutoNom software (MDL, San Leandro,Calif.). For example, compounds of formula I wherein W is O havetypically been named as ester derivatives of biphenyl-2-ylcarbamic acid.

REPRESENTATIVE EMBODIMENTS

The following substituents and values are intended to providerepresentative examples of various aspects and embodiments of thisinvention. These representative values are intended to further defineand illustrate such aspects and embodiments and are not intended toexclude other embodiments or to limit the scope of this invention. Inthis regard, the representation that a particular value or substituentis preferred is not intended in any way to exclude other values orsubstituents from this invention unless specifically indicated.

The values for a and b are independently 0, 1, 2, 3, 4 or 5;particularly independently 0, 1 or 2, and even more particularly 0 or 1.In one embodiment, both a and b are 0.

When present, each R¹ may be at the 2, 3, 4, 5 or 6-position of thephenyl ring to which it is attached. Each R¹ is independently selectedfrom (1-4C)alkyl, (2-4C)alkenyl, (2-4C)alkynyl, (3-6C)cycloalkyl, cyano,halo, —OR^(1a), —C(O)OR^(1b), —SR^(1c), —S(O)R^(1d), —S(O)₂R^(1e),—NR^(1f)R^(1g), —NR^(1h)S(O)₂R^(1i), and —NR^(1j)C(O)R^(1k), examples ofwhich include methyl, fluoro, chloro, bromo, hydroxy, methoxy, amino,methylamino, dimethylamino and the like. Particular values for R¹ arefluoro or chloro.

When present, each R² may be at the 3, 4, 5 or 6-position on thephenylene ring to which it is attached (where the carbon atom on thephenylene ring attached to the nitrogen atom is position 1). Each R² isindependently selected from (1-4C)alkyl, (2-4C)alkenyl, (24C)alkynyl,(3-6C)cycloalkyl, cyano, halo, —OR^(2a), —C(O)OR^(2b), —SR^(2c),—S(O)R^(2d), —S(O)₂R^(2e), —NR^(2f)R^(2g), —NR^(2h)S(O)₂R^(2i), and—NR^(2j)C(O)R^(2k), examples of which include methyl, fluoro, chloro,bromo, hydroxy, methoxy, amino, methylamino, dimethylamino and the like.Particular values for R² are fluoro or chloro.

Each R^(1a), R^(1b), R^(1c), R^(1d), R^(1e), R^(1f), R^(1g), R^(1h),R^(1i), R^(1j), and R^(1k) and R^(2a), R^(2b), R^(2c), R^(2d), R^(2e),R^(2f), R^(2g), R^(2h), R^(2i), R^(2j), and R^(2k) as used in R¹ and R²,respectively, is independently hydrogen, (1-4C)alkyl orphenyl(1-4C)alkyl, examples of which include hydrogen, methyl, ethyl,n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl or benzyl.In one embodiment, these groups are independently hydrogen or(1-3C)alkyl. In another embodiment, these groups are independentlyhydrogen, methyl or ethyl. In addition, each alkyl and alkoxy group inR¹, R^(1a-1k), R², and R^(2a-2k) is optionally substituted with 1 to 5fluoro substituents.

In one embodiment of this invention, W is O. In another embodiment, W isNW^(a). Generally, it has been found that compounds in which Wrepresents O exhibit particularly high affinity for muscarinicreceptors. Accordingly, in a particular embodiment of this invention, Wrepresents O.

When W is NW^(a), W^(a) is hydrogen or (1-4C)alkyl, examples of whichinclude hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl,sec-butyl, isobutyl and tert-butyl. In one embodiment, W^(a) is hydrogenor (1-3C)alkyl. In another embodiment, W^(a) is hydrogen, methyl orethyl, particularly hydrogen or methyl. In yet another embodiment, W^(a)is hydrogen and NW^(a) is NH.

The value for c is 0, 1, 2, 3, 4, or 5; particularly 0, 1, or 2; andmore particularly 0 or 1. In one particular embodiment, c is 0. Inanother embodiment, c is 2.

Each R³ independently represents (1-4C)alkyl or two R³ groups that arejoined to form (1-3C)alkylene, (2-3C)alkenylene or oxiran-2,3-diyl. Inone embodiment, each R³ is independently (t-4C)alkyl, such as methyl,ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl and tert-butyl.In addition, each alkyl group in R³ is optionally substituted with 1 to5 fluoro substituents. In one embodiment, each R³ is independently(1-3C)alkyl, and in another embodiment, each R³ is independently methylor ethyl.

In one embodiment, each R³ is at the 3, 4 or 5-position on thepiperidine ring (where the nitrogen atom of the piperidine ring isposition 1). In a particular embodiment, R³ is at the 4-position on thepiperidine ring. In another embodiment, R³ is at the 1-position of thepiperidine ring, i.e., on the nitrogen atom of the piperidine ring thusforming a quaternary amine salt.

In yet another embodiment, two R³ groups are joined to form a(1-3C)alkylene or (2-3C)alkenylene group. For example, two R³ groups atthe 2 and 6-positions on the piperidine ring can be joined to form anethylene bridge (i.e., the piperidine ring and the R³ groups form an8-azabicyclo[3.2.1]octane ring); or two R³ groups at the 1 and4-positions on the piperidine ring can be joined to form an ethylenebridge (i.e., the piperidine ring and the R³ groups form an1-azabicyclo[2.2.2]octane ring). In this embodiment, other R³ groups asdefined herein may also be present.

In still another embodiment, two R³ groups are joined to form aoxiran-2,3-diyl group. For example, two R³ groups at the 2 and6-positions on the piperidine ring can be joined to form a3-oxatricyclo[3.3.1.0^(2,4)]nonane ring). In this embodiment, other R³groups as defined herein may also be present.

The value for m is 0 or 1. In one embodiment, m is 0.

R⁴ represents hydrogen, (1-4C)alkyl, or (3-4C)cycloalkyl. Examplesof(1-4C)alkyl include methyl, ethyl, n-propyl, isopropyl, n-butyl,sec-butyl, isobutyl and tert-butyl. Examples of (3-4C)cycloalkyl groupsinclude cyclopropyl and cyclobutyl. In one embodiment R⁴ representshydrogen or (1-3C)alkyl, in particular hydrogen, methyl or ethyl. Inanother embodiment, R⁴ is hydrogen.

The value for s is 0, 1 or 2. A particular value for s is 0 or 1. In oneembodiment, s is 0. In another embodiment, s is 2.

Ar¹ is a phenylene group or a (3-5C)heteroarylene group containing 1 or2 heteroatoms independently selected from oxygen, nitrogen or sulfur.The phenylene or heteroarylene group may be unsubstituted (q is 0) orsubstituted with 1, 2, 3, or 4 (q is 1, 2, 3, or 4) R⁵ substituents,which are independently selected from halo, hydroxy, (1-4C)alkyl or(1-4C)alkoxy. In addition, each alkyl and alkoxy group in R⁵ isoptionally substituted with 1 to 5 fluoro substituents. The value for qis 0, 1, 2, 3, or 4, particularly 0, 1, 2 or 3. In one embodiment, q is0, 1 or 2. The point of attachment for Ar¹ is at any available carbon orheteroatom ring atom. In certain embodiments, Ar¹ is a phenylene groupattached at the meta or para position.

In one embodiment Ar¹ is phen-1,3-ylene or phen-1,4-ylene wherein thephenylene group is unsubstituted or substituted with 1, 2 or 3 R⁵substituents. Representative R⁵ substituents include fluoro, chloro,bromo, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl,tert-butyl, methoxy, ethoxy, isopropoxy, difluoromethyl,trifluoromethyl, 2,2,2-trifluoroethyl and trifluoromethoxy. Particularexamples of Ar¹ groups in this embodiment include2-fluorophen-1,4-ylene, 3-fluorophen-1,4-ylene, 2-chlorophen-1,4-ylene,3-chlorophen-1,4-ylene, 2-methylphen-1,4-ylene, 3-methylphen-1,4-ylene,2-methoxyphen-1,4-ylene, 3-methoxyphen-1,4-ylene,2-trifluoromethoxyphen-1,4-ylene, 3-trifluoromethoxyphen-1,4-ylene,2,3-difluorophen-1,4-ylene, 2,5-difluorophen-1,4-ylene,2,6-difluorophen-1,4-ylene, 2,3-dichlorophen-1,4-ylene,2,5-dichlorophen-1,4-ylene, 2,6-dichlorophen-1,4-ylene,2-chloro-5-methoxyphen-1,4-ylene, 2-chloro-6-methoxyphen-1,4-ylene,2-chloro-5-trifluoromethoxyphen-1,4-ylene,2-chloro-6-trifluoromethoxyphen-1,4-ylene, and2,5-dibromophen-1,4-ylene.

In another embodiment, Ar¹ is a (3-5C)heteroarylene group containing 1or 2 heteroatoms independently selected from oxygen, nitrogen or sulfur;wherein the heteroarylene group is unsubstituted or substituted with 1or 2 R⁵ substituents. Representative heteroarylene groups includedivalent species of pyrrole, imidazole, thiazole, oxazole, furan,thiophene, pyrazole, isoxazole, isothiazole, pyridine, pyrazine,pyridazine and pyrimidine, where the point of attachment is at anyavailable carbon or nitrogen ring atom. More specific examples of suchAr¹ groups include 2,5-furylene, 2,4-thienylene, 2,5-thienylene,2,5-pyridylene, 2,6-pyridylene, and 2,5-pyrrolylene. Representative R⁵substituents include fluoro, chloro, methyl, ethyl, n-propyl, isopropyl,n-butyl, isobutyl, sec-butyl, tert-butyl, methoxy, ethoxy, isopropoxy,difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl andtrifluoromethoxy. Particular examples of substituted Ar¹ groups include3-fluoro-2,5-thienylene, 3-chloro-2,5-thienylene,3-methyl-2,5-thienylene, 3-methoxy-2,5-thienylene, and3-methoxy-6-chloro-2,5-pyridylene.

In one particular embodiment, Ar¹ represents phen-1,3-ylene,phen-1,4-ylene, 2,4-thienylene or 2,5-thienylene; wherein the phenyleneor thienylene group is optionally substituted with 1 or 2 R⁵substituents. In another particular embodiment, Ar¹ representsphen-1,4-ylene or 2,4-thienylene optionally substituted with 1 or 2 R⁵substituents.

The value for t is 0, 1 or 2. A particular value for t is 1.

The value for n is 0, 1, 2, or 3. Particular values for n are 1 or 2. Inone embodiment, n is 2.

The value for d is 0, 1, 2, 3, or 4. Particular values for d are 0, 1 or2. In one embodiment, d is 0.

Each R⁶ independently represents fluoro or (1-4C)alkyl, examples ofwhich include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl,isobutyl and tert-butyl. In addition, each alkyl and alkoxy group in R⁶is optionally substituted with 1 to 5 fluoro substituents. In oneembodiment, each R⁶ independently represents fluoro or (1-3C)alkyl, andin another embodiment, each R⁶ is independently selected from fluoro,methyl, ethyl or trifluoromethyl.

The value for p is 0 or 1. In one particular embodiment, p is 0.

R⁷ and R⁸ each independently represent hydrogen or (1-4C)alkyl, examplesof which include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl,isobutyl and tert-butyl. In one embodiment, R⁷ and R⁸ each independentlyrepresent hydrogen or (1-3C)alkyl. In a particular embodiment, R⁷ ishydrogen, methyl, ethyl, n-propyl or isopropyl, and R⁸ is hydrogen. Inanother particular embodiment, R⁷ and R⁸ are both hydrogen or bothethyl. In addition, each alkyl and alkoxy group in R⁷ and R⁸ isoptionally substituted with 1 to 5 fluoro substituents.

As noted in formula I, the —CONR⁷R⁸ group can be located at any carbonatom on the ring. For example, when n is 2, the —CONR⁷R⁸ group can belocated at the ortho, meta or para position. In one embodiment, the—CONR⁷R⁸ group is located at the meta or para position; and in aparticular embodiment, the —CONR⁷R⁸ group is located at the paraposition.

A particular group of compounds of interest are compounds of formula Iwherein a, b, c and d are 0; n is 2; and R⁴ is hydrogen, methyl orethyl.

Another particular group of compounds of interest are compounds offormula I wherein a, b, c and d are 0; R⁴ is hydrogen, methyl or ethyl;and R⁷ is hydrogen.

Another particular group of compounds of interest are compounds offormula I wherein a, b, c and d are 0; R⁴ is hydrogen, methyl or ethyl;R⁷ is hydrogen, methyl, ethyl, n-propyl or isopropyl, and R⁸ ishydrogen.

Another particular group of compounds of interest are compounds offormula I wherein a, b, c and d are 0; R⁴ is hydrogen, methyl or ethyl;and R⁷ and R⁸ are ethyl.

Another particular group of compounds of interest are compounds offormula I wherein a, b, c and d are 0; R⁴ is hydrogen, methyl or ethyl;R⁷ and R⁸ are hydrogen; and s is 0.

Another particular group of compounds of interest are compounds offormula I wherein a, b, c and d are 0; R⁴ is hydrogen, methyl or ethyl;R⁷ and R⁸ are hydrogen; s is 0; and t is 1.

Another particular group of compounds of interest are compounds offormula I wherein a, b, c and d are 0; R⁴ is hydrogen, methyl or ethyl;R⁷ and R⁸ are hydrogen; s is 0; t is 1; and m is 0.

Representative Subgeneric Groupings

The following subgeneric formulae and groupings are intended to providerepresentative examples of various aspects and embodiments of thisinvention and as such, they are not intended to exclude otherembodiments or to limit the scope of this invention unless otherwiseindicated.

A particular group of compounds of formula I are those disclosed in U.S.Provisional Application No. 60/552,443, filed on Mar. 11, 2004. Thisgroup includes compounds of formula Ia:

wherein:

a is 0 or an integer of from 1 to 3; each R¹ is independently selectedfrom (1-4C)alkyl, (2-4C)alkenyl, (2-4C)alkynyl, (3-6C)cycloalkyl, cyano,halo, —OR^(1a), —C(O)OR^(1b), —SR^(1c), —S(O)R^(1d), —S(O)₂R^(1e) and—NR^(1f)R^(1g); each of R^(1a), R^(1b), R^(1c), R^(1d), R^(1e), R^(1f)and R^(1g) is independently hydrogen, (1-4C)alkyl or phenyl(t-4C)alkyl;

b is 0 or an integer of from 1 to 3; each R² is independently selectedfrom (1-4C)alkyl, (2-4C)alkenyl, (2-4C)alkynyl, (3-6C)cycloalkyl, cyano,halo, —OR^(2a) —C(O)OR^(2b), —SR^(2c), S(O)R^(2d), —S(O)₂R^(2e) and—NR^(2f)R^(2g); each of R^(2a), R^(2b), R^(2c), R^(2d), R^(2e), R^(2f)and R^(2g) is independently hydrogen, (1-4C)alkyl or phenyl(1-4C)alkyl;

W represents O or NW^(a), where W^(a) is hydrogen or (1-4C)alkyl;

c is 0 or an integer from 1 to 4; each R³ independently represents(1-4C)alkyl;

m is 0 or 1;

R⁴ is hydrogen or (1-4C)alkyl;

s is 0 or 1;

Ar¹ represents a phenylene group or a (3-5C)heteroarylene groupcontaining 1 or 2 heteroatoms selected independently from oxygen,nitrogen or sulfur; wherein the phenylene or heteroarylene group issubstituted with (R⁵)_(q) where q is 0 or an integer from 1 to 4 andeach R⁵ is selected independently from halo, hydroxy, (1-4C)alkyl or(1-4C)alkoxy;

t is 0 or 1;

n is 0, 1 or 2;

d is 0 or an integer from 1 to 4; each R⁶ independently representsfluoro or (1-4C)alkyl; and

R⁷ is hydrogen or (1-4C)alkyl;

wherein each alkyl and alkoxy group in R¹, R^(1a-1g), R², R^(2a-2g), R³,R⁵, R⁶ or R⁷ is optionally substituted with 1 to 5 fluoro substituents;or a pharmaceutically acceptable salt or solvate or stereoisomerthereof.

This group also includes compounds of formula Ib:

wherein: R⁴, q, R⁵ and R⁷ are as defined for formula Ia; or apharmaceutically acceptable salt or solvate or stereoisomer thereof. Aparticular embodiment includes compounds of formula Ib, where q is 0, 1or 2, and R⁵ is independently selected from halo, (1-4C)alkyl or(1-4C)alkoxy, wherein each alkyl and alkoxy group is optionallysubstituted with from 1 to 3 fluoro substituents.

In addition, particular compounds of formula I that are of interestinclude:

-   biphenyl-2-ylcarbamic acid    1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-yl    ester;-   biphenyl-2-ylcarbamic acid    1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]ethylamino I    ethyl)piperidin-4-yl ester;-   biphenyl-2-ylcarbamic acid    1-(2-{methyl-[4-(4-methylcarbamoylpiperidin-1-ylmethyl)benzoyl]amino}ethyl)piperidin-4-yl    ester;-   biphenyl-2-ylcarbamic acid    1-(2-{[4-(4-ethylcarbamoylpiperidin-1-ylmethyl)    benzoyl]methylamino}ethyl)piperidin-4-yl ester;-   biphenyl-2-ylcarbamic acid    1-(2-{methyl-[4-(4-propylcarbamoylpiperidin-1-ylmethyl)benzoyl]amino}ethyl)piperidin-4-yl    ester;-   biphenyl-2-ylcarbamic acid    1-(2-{[4-(4-isopropylcarbamoylpiperidin-1-ylmethyl)    benzoyl]methylamino}ethyl)piperidin-4-yl ester;-   biphenyl-2-ylcarbamic acid    1-{2-[4-(4-carbamoylpiperidin-1-ylmethyl)-2-fluorobenzoylamino]ethyl}piperidin-4-yl    ester;-   biphenyl-2-ylcarbamic acid    1-(2-{[2,5-d]bromo-4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-yl    ester;-   biphenyl-2-ylcarbamic acid    1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)-2-fluorobenzoyl]methylamino}ethyl)piperidin-4-yl    ester;-   biphenyl-2-ylcarbamic acid    1-{2-[4-(4-diethylcarbamoylpiperidin-1-ylmethyl)-2-fluorobenzoylamino]ethyl}piperidin-4-yl    ester;-   biphenyl-2-ylcarbamic acid    1-(2-{[4-(4-diethylcarbamoylpiperidin-1-ylmethyl)-2-fluorobenzoyl]methylamino}ethyl)piperidin-4-yl    ester;-   biphenyl-2-ylcarbamic acid    1-(2-{[4-(4-diethylcarbamoylpiperidin-1-ylmethyl)    benzoyl]methylamino}ethyl)piperidin-4-yl ester;-   biphenyl-2-ylcarbamic acid    1-(2-{[4-(3-(S)-diethylcarbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-yl    ester;-   biphenyl-2-yl-carbamic acid    1-(2-{[4-(2-carbamoyl-piperidin-1-ylmethyl)    benzoyl]methylamino}ethyl)piperidinyl ester;-   biphenyl-2-yl-carbamic acid    1-(2-{[4-(4-carbamoyl-piperidin-1-ylmethyl)-2-methoxybenzoyl]methylamino}ethyl)piperidin-4-yl    ester;-   biphenyl-2-yl-carbamic acid    1-(2-{[5-(4-carbamoylpiperidin-1-ylmethyl)thiophene-2-carbonyl]methylamino}ethyl)piperidin-4-yl    ester;-   biphenyl-2-yl-carbamic acid    1-(2-{[5-((R)-3-diethylcarbamoylpiperidin-1-ylmethyl)thiophene-2-carbonyl]methylamino}ethyl)piperidin-4-yl    ester;-   biphenyl-2-yl-carbamic acid    1-(2-{[5-((R)-3-diethylcarbamoylpiperidin-1-ylmethyl)thiophene-2-carbonyl]amino}ethyl)piperidin-4-yl    ester;-   biphenyl-2-yl-carbamic acid    1-(2-{[5-(4-carbamoylpiperidin-1-ylmethyl)thiophene-2-carbonyl]amino}ethyl)piperidin-4-yl    ester;-   biphenyl-2-yl-carbamic acid    1-(2-{[5-((R)-3-diethylcarbamoylpiperidin-1-ylmethyl)-1H-pyrrole-2-carbonyl]methylamino}ethyl)piperidin-4-yl    ester,-   biphenyl-2-yl-carbamic acid    1-(2-{[5-(4-carbamoylpiperidin-1-ylmethyl)-1H-pyrrole-2-carbonyl]methylamino}ethyl)piperidin-4-yl    ester;-   biphenyl-2-yl-carbamic acid    1-(2-{[5-((R)-3-diethylcarbamoylpiperidin-1-ylmethyl)furan-2-carbonyl]methylamino}ethyl)piperidin-4-yl    ester;-   biphenyl-2-yl-carbamic acid    1-(2-{[5-(4-diethylcarbamoyl-piperidin-1-ylmethyl)furan-2-carbonyl]methylamino}ethyl)piperidin-4-yl    ester;-   biphenyl-2-yl-carbamic acid    1-(2-{[(5-(4-carbamoylpiperidin-1-ylmethyl)furan-2-carbonyl]-amino}ethyl)piperidin-4-yl    ester;-   biphenyl-2-yl-carbamic acid    1-(2-{[5-((R)-3-diethylcarbamoylpiperidin-1-ylmethyl)furan-2-carbonyl]amino}ethyl)piperidin-4-yl    ester;-   biphenyl-2-yl-carbamic acid    1-[2-({3-[4-(3-carbamoylpiperidin-1-ylmethyl)phenyl]propionyl}    methylamino)ethyl]piperidin-4-yl ester;-   biphenyl-2-yl-carbamic acid    1-[2-({3-[4-(4-carbamoylpiperidin-1-ylmethyl)phenyl]propionyl}methylamino)ethyl]piperidin-4-yl    ester;-   biphenyl-2-yl-carbamic acid    1-(2-{3-[4-(4-carbamoylpiperidin-1-ylmethyl)phenyl]propionylamino}ethyl)piperidin-4-yl    ester;-   biphenyl-2-yl-carbamic acid    1-(2-{3-[4-(4-diethylcarbamoylpiperidin-1-ylmethyl)phenyl]propionylamino}ethyl)piperidin-4-yl    ester;-   biphenyl-2-yl-carbamic acid    1-(2-{3-[4-(3-diethylcarbamoylpiperidin-1-ylmethyl)phenyl]propionylamino}ethyl)piperidin-4-yl    ester;-   biphenyl-2-ylcarbamic acid 1-{2-[4-(4-carbamoylpiperidin-1-ylmethyl)    benzoylamino]ethyl}piperidin-4-yl ester;-   biphenyl-2-ylcarbamic acid    1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)-2-chloro-benzoyl]methylamino}ethyl)piperidin-4-yl    ester;-   biphenyl-2-ylcarbamic acid    1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)-2-chloro-5-methoxybenzoyl]methylamino}ethyl)piperidin-4-yl    ester; and-   biphenyl-2-ylcarbamic acid    1-[2-({2-[4-(4-carbamoylpiperidin-1-ylmethyl)phenyl]acetyl}methylamino)ethyl]piperidin-4-yl    ester;-   or a pharmaceutically acceptable salt or solvate thereof.    Definitions

When describing the compounds, compositions, methods and processes ofthis invention, the following terms have the following meanings unlessotherwise indicated.

The term “alkyl” means a monovalent saturated hydrocarbon group whichmay be linear or branched. Unless otherwise defined, such alkyl groupstypically contain from 1 to 10 carbon atoms. Representative alkyl groupsinclude, by way of example, methyl, ethyl, n-propyl, isopropyl, n-butyl,sec-butyl, isobutyl, tert-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl,n-nonyl, n-decyl and the like.

The term “alkylene” means a divalent saturated hydrocarbon group whichmay be linear or branched. Unless otherwise defined, such alkylenegroups typically contain from 1 to 10 carbon atoms. Representativealkylene groups include, by way of example, methylene, ethane-1,2-diyl(“ethylene”), propane-1,2-diyl, propane-1,3-diyl, butane-1,4-diyl,pentane-1,5-diyl and the like.

The term “alkoxy” means a monovalent group of the formula (alkyl)-O—,where alkyl is as defined herein. Representative alkoxy groups include,by way of example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy,sec-butoxy, isobutoxy, tert-butoxy and the like.

The term “alkenyl” means a monovalent unsaturated hydrocarbon groupwhich may be linear or branched and which has at least one, andtypically 1, 2 or 3, carbon-carbon double bonds. Unless otherwisedefined, such alkenyl groups typically contain from 2 to 10 carbonatoms. Representative alkenyl groups include, by way of example,ethenyl, n-propenyl, isopropenyl, n-but-2-enyl, n-hex-3-enyl and thelike. The term “alkenylene” means a divalent alkenyl group.

The term “alkynyl” means a monovalent unsaturated hydrocarbon groupwhich may be linear or branched and which has at least one, andtypically 1, 2 or 3, carbon-carbon triple bonds. Unless otherwisedefined, such alkynyl groups typically contain from 2 to 10 carbonatoms. Representative alkynyl groups include, by way of example,ethynyl, n-propynyl, n-but-2-ynyl, n-hex-3-ynyl and the like. The term“alkynylene” means a divalent alkynyl group.

The term “aryl” means a monovalent aromatic hydrocarbon having a singlering (i.e., phenyl) or fused rings (i.e., naphthalene). Unless otherwisedefined, such aryl groups typically contain from 6 to 10 carbon ringatoms. Representative aryl groups include, by way of example, phenyl andnaphthalene-1-yl, naphthalene-2-yl, and the like. The term “arylene”means a divalent aryl group.

The term “azacycloalkyl” means a monovalent heterocyclic ring containingone nitrogen atom, i.e., a cycloalkyl group in which one carbon atom hasbeen replaced with a nitrogen atom. Unless otherwise defined, suchazacycloalkyl groups typically contain from 2 to 9 carbon atoms.Representative examples of an azacycloalkyl group are pyrrolidinyl andpiperidinyl groups. The term “azacycloalkylene” means a divalentazacycloalkyl group. Representative examples of an azacycloalkylenegroup are pyrrolidinylene and piperidinylene groups.

The term “cycloalkyl” means a monovalent saturated carbocyclichydrocarbon group. Unless otherwise defined, such cycloalkyl groupstypically contain from 3 to 10 carbon atoms. Representative cycloalkylgroups include, by way of example, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl and the like. The term “cycloalkylene” means a divalentcycloalkyl group.

The term “halo” means fluoro, chloro, bromo and iodo.

The term “heteroaryl” means a monovalent aromatic group having a singlering or two fused rings and containing in the ring at least oneheteroatom (typically 1 to 3 heteroatoms) selected from nitrogen, oxygenor sulfur. Unless otherwise defined, such heteroaryl groups typicallycontain from 5 to 10 total ring atoms. Representative heteroaryl groupsinclude, by way of example, monovalent species of pyrrole, imidazole,thiazole, oxazole, furan, thiophene, triazole, pyrazole, isoxazole,isothiazole, pyridine, pyrazine, pyridazine, pyrimidine, triazine,indole, benzofuran, benzothiophene, benzimidazole, benzthiazole,quinoline, isoquinoline, quinazoline, quinoxaline and the like, wherethe point of attachment is at any available carbon or nitrogen ringatom. The term “heteroarylene” means a divalent heteroaryl group.

The term “heterocyclyl” or “heterocyclic” means a monovalent saturatedor unsaturated (non-aromatic) group having a single ring or multiplecondensed rings and containing in the ring at least one heteroatom(typically 1 to 3 heteroatoms) selected from nitrogen, oxygen or sulfur.Unless otherwise defined, such heterocyclic groups typically containfrom 2 to 9 total ring carbon atoms. Representative heterocyclic groupsinclude, by way of example, monovalent species of pyrrolidine,imidazolidine, pyrazolidine, piperidine, 1,4-dioxane, morpholine,thiomorpholine, piperazine, 3-pyrroline and the like, where the point ofattachment is at any available carbon or nitrogen ring atom. The term“heterocyclene” means a divalent heterocyclyl or heterocyclic group.

When a specific number of carbon atoms is intended for a particular termused herein, the number of carbon atoms is shown in parenthesespreceding the term. For example, the term “(1-4C)alkyl” means an alkylgroup having from 1 to 4 carbon atoms.

The term “pharmaceutically acceptable salt” means a salt which isacceptable for administration to a patient, such as a mammal (e.g.,salts having acceptable mammalian safety for a given dosage regime).Such salts can be derived from pharmaceutically acceptable inorganic ororganic bases and from pharmaceutically acceptable inorganic or organicacids. Salts derived from pharmaceutically acceptable inorganic basesinclude ammonium, calcium, copper, ferric, ferrous, lithium, magnesium,manganic, manganous, potassium, sodium, zinc and the like. Particularlypreferred are ammonium, calcium, magnesium, potassium and sodium salts.Salts derived from pharmaceutically acceptable organic bases includesalts of primary, secondary and tertiary amines, including substitutedamines, cyclic amines, naturally-occurring amines and the like, such asarginine, betaine, caffeine, choline, N,N′-dibenzylethylenediamine,diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol,ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine,glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine,methylglucamine, morpholine, piperazine, piperadine, polyamine resins,procaine, purines, theobromine, triethylamine, trimethylamine,tripropylamine, tromethamine and the like. Salts derived frompharmaceutically acceptable acids include acetic, ascorbic,benzenesulfonic, benzoic, camphosulfonic, citric, ethanesulfonic,edisylic, fumaric, gentisic, gluconic, glucoronic, glutamic, hippuric,hydrobromic, hydrochloric, isethionic, lactic, lactobionic, maleic,malic, mandelic, methanesulfonic, mucic, naphthalenesulfonic,naphthalene-1,5-disulfonic, naphthalene-2,6-disulfonic, nicotinic,nitric, orotic, pamoic, pantothenic, phosphoric, succinic, sulfuric,tartaric, p-toluenesulfonic, xinafoic and the like. Particularlypreferred are citric, hydrobromic, hydrochloric, isethionic, maleic,naphthalene-1,5-disulfonic, phosphoric, sulfuric and tartaric acids.

The term “salt thereof” means a compound formed when the hydrogen of anacid is replaced by a cation, such as a metal cation or an organiccation and the like. Preferably, the salt is a pharmaceuticallyacceptable salt, although this is not required for salts of intermediatecompounds that are not intended for administration to a patient.

The term “solvate” means a complex or aggregate formed by one or moremolecules of a solute, i.e. a compound of formula I or apharmaceutically acceptable salt thereof, and one or more molecules of asolvent. Such solvates are typically crystalline solids having asubstantially fixed molar ratio of solute and solvent. Representativesolvents include, by way of example, water, methanol, ethanol,isopropanol, acetic acid and the like. When the solvent is water, thesolvate formed is a hydrate.

It will be appreciated that the term “or a pharmaceutically acceptablesalt or solvate or stereoisomer thereof” is intended to include allpermutations of salts, solvates and stereoisomers, such as a solvate ofa pharmaceutically acceptable salt of a stereoisomer of a compound offormula I.

The term “therapeutically effective amount” means an amount sufficientto effect treatment when administered to a patient in need of treatment.

The term “treating” or “treatment” as used herein means the treating ortreatment of a disease or medical condition (such as COPD) in a patient,such as a mammal (particularly a human) that includes:

-   -   (a) preventing the disease or medical condition from occurring,        i.e., prophylactic treatment of a patient;    -   (b) ameliorating the disease or medical condition, i.e.,        eliminating or causing regression of the disease or medical        condition in a patient;    -   (c) suppressing the disease or medical condition, i.e., slowing        or arresting the development of the disease or medical condition        in a patient; or    -   (d) alleviating the symptoms of the disease or medical condition        in a patient.

The term “leaving group” means a functional group or atom which can bedisplaced by another functional group or atom in a substitutionreaction, such as a nucleophilic substitution reaction. By way ofexample, representative leaving groups include chloro, bromo and iodogroups; sulfonic ester groups, such as mesylate, tosylate, brosylate,nosylate and the like; and acyloxy groups, such as acetoxy,trifluoroacetoxy and the like.

The term “protected derivatives thereof” means a derivative of thespecified compound in which one or more functional groups of thecompound are protected from undesired reactions with a protecting orblocking group. Functional groups which may be protected include, by wayof example, carboxylic acid groups, amino groups, hydroxyl groups, thiolgroups, carbonyl groups and the like. Representative protecting groupsfor carboxylic acids include esters (such as a p-methoxybenzyl ester),amides and hydrazides; for amino groups, carbamates (such astert-butoxycarbonyl) and amides; for hydroxyl groups, ethers and esters;for thiol groups, thioethers and thioesters; for carbonyl groups,acetals and ketals; and the like. Such protecting groups are well-knownto those skilled in the art and are described, for example, in T. W.Greene and G. M. Wuts, Protecting Groups in Organic Synthesis, ThirdEdition, Wiley, New York, 1999, and references cited therein.

The term “amino-protecting group” means a protecting group suitable forpreventing undesired reactions at an amino group. Representativeamino-protecting groups include, but are not limited to,tert-butoxycarbonyl (BOC), trityl (Tr), benzyloxycarbonyl (Cbz),9-fluorenylmethoxycarbonyl (Fmoc), formyl, trimethylsilyl (TMS),tert-butyldimethylsilyl (TBS), and the like.

The term “carboxy-protecting group” means a protecting group suitablefor preventing undesired reactions at a carboxy group. Representativecarboxy-protecting groups include, but are not limited to, esters, suchas methyl, ethyl, tert-butyl, benzyl (Bn), p-methoxybenzyl (PMB),9-fluorenylmethyl (Fm), trimethylsilyl (TMS), tert-butyldimethylsilyl(TBS), diphenylmethyl (benzhydryl, DPM) and the like.

The term “hydroxyl-protecting group” means a protecting group suitablefor preventing undesirable reactions at a hydroxyl group. Representativehydroxyl-protecting groups include, but are not limited to, silyl groupsincluding tri(1-6C)alkylsilyl groups, such as trimethylsilyl (TMS),triethylsilyl (TES), tert-butyldimethylsilyl (TBS) and the like; esters(acyl groups) including (1-6C)alkanoyl groups, such as formyl, acetyland the like; arylmethyl groups, such as benzyl (Bn), p-methoxybenzyl(PMB), 9-fluorenylmethyl (Fm), diphenylmethyl (benzhydryl, DPM) and thelike. Additionally, two hydroxyl groups can also be protected as analkylidene group, such as prop-2-ylidine, formed, for example, byreaction with a ketone, such as acetone.

General Synthetic Procedures

The biphenyl compounds of this invention can be prepared from readilyavailable starting materials using the following general methods andprocedures or by using other information readily available to those ofordinary skill in the art. Although a particular embodiment of thepresent invention may be shown or described herein, those skilled in theart will recognize that all embodiments or aspects of the presentinvention can be prepared using the methods described herein or by usingother methods, reagents and starting materials known to those skilled inthe art. It will also be appreciated that where typical or preferredprocess conditions (i.e., reaction temperatures, times, mole ratios ofreactants, solvents, pressures, etc.) are given, other processconditions can also be used unless otherwise stated. While the optimumreaction conditions may vary depending on the particular reactants orsolvent used, such conditions can be readily determined by one skilledin the art by routine optimization procedures.

Additionally, as will be apparent to those skilled in the art,conventional protecting groups may be necessary or desired to preventcertain functional groups from undergoing undesired reactions. Thechoice of a suitable protecting group for a particular functional groupas well as suitable conditions for protection and deprotection of suchfunctional groups are well-known in the art. Protecting groups otherthan those illustrated in the procedures described herein may be used,if desired. For example, numerous protecting groups, and theirintroduction and removal, are described in T. W. Greene and G. M. Wuts,Protecting Groups in Organic Synthesis, Third Edition, Wiley, New York,1999, and references cited therein.

By way of illustration, the compounds of formula I can be prepared by aprocess comprising:

(a) reacting a compound of formula II:

or a salt thereof, with a compound of formula III:

wherein Z¹ represents a leaving group; or

(b) coupling a compound of formula IV:

with a compound of formula V:

or a reactive derivative thereof; or

(c) reacting a compound of formula VI:

wherein Z² represents a leaving group; with a compound of formula VII:

or

(d) reacting a compound of formula II with a compound of formula VIII:

in the presence of a reducing agent; or

(e) reacting a compound of formula IX:

with a compound of formula VII in the presence of a reducing agent; or

(f) reacting a compound of formula XVIII:

where R′ is H, —CH₃ or —CH₂CH₃, with a compound of formula XIX:NHR⁷R⁸  XIXand then

(g) removing any protecting groups that may be present to provide acompound of formula I; and optionally, forming a pharmaceuticallyacceptable salt thereof.

Generally, if a salt of one of the starting materials is used in theprocesses described above, such as an acid addition salt, the salt istypically neutralized before or during the reaction process. Thisneutralization reaction is typically accomplished by contacting the saltwith one molar equivalent of a base for each molar equivalent of acidaddition salt.

In process (a), the reaction between the compounds of formula II andIII, the leaving represented by Z¹ can be, for example, halo, such aschloro, bromo or iodo, or a sulfonic ester group, such as mesylate ortosylate. The reaction is conveniently performed in the presence of abase, for example, a tertiary amine such as diisopropylethylamine.Convenient solvents include nitrites, such as acetonitrile. The reactionis conveniently conducted at a temperature in the range of from 0° C. to100° C.

Compounds of formula II are generally known in the art, or can beprepared by deprotecting a compound of formula X:

wherein P¹ represents an amino-protecting group, such as a benzyl group.Benzyl groups are conveniently removed by reduction, using a hydrogen orammonium formate and a Group VIII metal catalyst, such as palladium.When W represents NW^(a), the hydrogenation is conveniently performedusing Pearlman's catalyst (Pd(OH)₂).

Compounds of formula X can be prepared by reacting an isocyanatecompound of formula XI:

with a compound of formula XII:

Compounds of formula III can be prepared starting from a correspondingcompound in which Z¹ represents a hydroxyl group, for example, byreaction of a halogenating agent, such as thionyl chloride, to afford acompound of formula III in which Z¹ represents halo, such as chloro.Compounds in which Z¹ represents a hydroxyl group may be prepared, forexample, by reacting a compound of formula V with an appropriateamino-substituted alcohol, such as 2-aminoethanol or 3-aminopropan-1-ol.

In process (b), a compound of formula IV is reacted with a compound offormula V or reactive derivative thereof. By “reactive derivative” ofcompound V, it is meant that the carboxylic acid is activated, forexample, by forming an anhydride or carboxylic acid halide, such as acarboxylic acid chloride. Alternatively, the carboxylic acid can beactivated using conventional carboxylic acid/amine coupling reagents,such carbodiimides,O-(7-azabenzotriazol-1-yl-N,N,N′,N′tetramethyluroniumhexafluorophosphate (HATU) and the like. This reaction is convenientlyperformed under conventional amide bond-forming conditions. The processis conveniently conducted at a temperature in the range of from −10° C.to 100° C.

Compounds of formula IV can be prepared by reacting a compound offormula II with a compound of formula XIII:OHC(CH₂)_(m)CH₂NR⁴P²  XIIIwherein P² represents hydrogen or an amino-protecting group, such asbenzyl, in the presence of a reducing agent, such as sodiumtriacetoxyborohydride, followed if necessary by removing theamino-protecting group P² by, for example, hydrogenation in the presenceof palladium.

Compounds of formula V can be prepared by reacting a compound of formulaVII with a compound of formula XIV:

wherein P³ represents hydrogen or a carboxyl-protecting group, such asmethyl or ethyl, and Z³ represents a leaving group, followed ifnecessary by removing the carboxyl protecting group P³. Alternatively,such compounds can be prepared by reductive amination of a compound offormula XV:

with a compound of formula VII under conventional reaction conditions,such as those described for processes (d) and (e).

Referring to process (c), the leaving group represented by Z² can be,for example, halo, such as chloro, bromo or iodo, or a sulfonic estergroup, such as mesylate or tosylate. This reaction is convenientlyperformed in the presence of a base, for example, a tertiary amine suchas diisopropylethylamine. Convenient solvents include nitrites, such asacetonitrile. The reaction is conveniently conducted at a temperature inthe range of from 0° C. to 100° C. The compounds of formula VI can beprepared by reacting a compound of formula IV with a compound of formulaXVI:

or a reactive derivative thereof, such as an acid chloride or anhydride.The reaction is conveniently performed following, for example, themethod of process (b) described herein. Compounds of formula VII aregenerally known or can be prepared from readily available startingmaterials using well-known synthetic methods.

In process (d), the reducing agent may be, for example, hydrogen in thepresence of a Group VIII metal catalyst, such as palladium, or a metalhydride reducing agent, such as a borohydride, including sodiumtriacetoxyborohydride. Convenient solvents include alcohols, such asmethanol. The reaction is conveniently performed at a temperature in therange of from 0° C. to 100° C. The compounds of formula VIII may beprepared by oxidizing a compound corresponding to formula III in whichZ¹ represents a hydroxyl group. Such oxidation reactions can beconducted, for example, using sulfur dioxide pyridine complex indimethylsulfoxide in the presence of a tertiary amine, such asdiisopropylethylamine.

In process (e), the reducing agent may be, for example, hydrogen in thepresence of a Group VIII metal catalyst, such as palladium, or a metalhydride reducing agent including borohydrides, such as sodiumtriacetoxyborohydride, optionally used in combination with a titaniumtetraalkoxide, such as titanium tetraisopropoxide. Convenient solventsinclude alcohols, such as methanol and halogenated hydrocarbons, such asdichloromethane. The reaction is conveniently performed at a temperaturein the range of from 0° C. to 100° C. Compounds of formula IX may beprepared by reacting a compound of formula IV with a compound of formulaXVII:

in the presence of a carboxylic acid/amine coupling agent, such as1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and1-hydroxybenzotriazole hydrate (HOBT) and the like.

Referring to process (f), compounds of formula XVIII may be prepared byreacting a compound of formula IX with a compound of formula VII in thepresence of a reducing agent, such as sodium triacetoxyborohydride,similar to that done in process (e).

As will be apparent to those skilled in the art, compounds of formula Iprepared by any of steps (a) to (f) herein may be further derivatized toform other compounds of formula I using methods and reagents well-knownin the art. By way of illustration, a compound of formula I may bereacted with bromine to afford a corresponding compound of formula I inwhich R², for example, represents a bromo group. Additionally, acompound of formula I in which R⁴ represents a hydrogen atom may bealkylated to afford a corresponding compound of formula I in which R⁴represents a (1-4C)alkyl group.

Certain of the intermediates described herein are believed to be noveland accordingly, such compounds are provided as further aspects of theinvention including, for example, the compounds of formula III, V, andVIII, and salts thereof.

Further details regarding specific reaction conditions and otherprocedures for preparing representative compounds of this invention orintermediates thereof are described in the Examples set forth below.

Pharmaceutical Compositions and Formulations

The biphenyl compounds of this invention are typically administered to apatient in the form of a pharmaceutical composition or formulation. Suchpharmaceutical compositions may be administered to the patient by anyacceptable route of administration including, but not limited to,inhaled, oral, nasal, topical (including transdermal) and parenteralmodes of administration.

It will be understood that any form of the compounds of this invention,(i.e., free base, pharmaceutically acceptable salt, solvate, etc.) thatis suitable for the particular mode of administration can be used in thepharmaceutical compositions discussed herein.

Accordingly, in one of its composition aspects, this invention isdirected to a pharmaceutical composition comprising a pharmaceuticallyacceptable carrier or excipient and a therapeutically effective amountof a compound of formula I, or a pharmaceutically acceptable salt orsolvate or stereoisomer thereof. Optionally, such pharmaceuticalcompositions may contain other therapeutic and/or formulating agents ifdesired.

The pharmaceutical compositions of this invention typically contain atherapeutically effective amount of a compound of the present inventionor a pharmaceutically acceptable salt or solvate or stereoisomerthereof. Typically, such pharmaceutical compositions will contain fromabout 0.01 to about 95% by weight of the active agent; including, fromabout 0.01 to about 30% by weight; such as from about 0.01 to about 10%by weight of the active agent.

Any conventional carrier or excipient may be used in the pharmaceuticalcompositions of this invention. The choice of a particular carrier orexcipient, or combinations of carriers or excipients, will depend on themode of administration being used to treat a particular patient or typeof medical condition or disease state. In this regard, the preparationof a suitable pharmaceutical composition for a particular mode ofadministration is well within the scope of those skilled in thepharmaceutical arts. Additionally, the ingredients for such compositionsare commercially available from, for example, Sigma, P.O. Box 14508, St.Louis, Mo. 63178. By way of further illustration, conventionalformulation techniques are described in Remington: The Science andPractice of Pharmacy, 20^(th) Edition, Lippincott Williams & White,Baltimore, Md. (2000); and H. C. Ansel et al., Pharmaceutical DosageForms and Drug Delivery Systems, 7^(th) Edition, Lippincott Williams &White, Baltimore, Md. (1999).

Representative examples of materials which can serve as pharmaceuticallyacceptable carriers include, but are not limited to, the following: (1)sugars, such as lactose, glucose and sucrose; (2) starches, such as cornstarch and potato starch; (3) cellulose, and its derivatives, such assodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate;(4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8)excipients, such as cocoa butter and suppository waxes; (9) oils, suchas peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil,corn oil and soybean oil; (10) glycols, such as propylene glycol; (11)polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol;(12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14)buffering agents, such as magnesium hydroxide and aluminum hydroxide;(15) alginic acid; (16) pyrogen-free water, (17) isotonic saline; (18)Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions;(21) compressed propellant gases, such as chlorofluorocarbons andhydrofluorocarbons; and (22) other non-toxic compatible substancesemployed in pharmaceutical compositions.

The pharmaceutical compositions of this invention are typically preparedby thoroughly and intimately mixing or blending a compound of theinvention with a pharmaceutically acceptable carrier and one or moreoptional ingredients. If necessary or desired, the resulting uniformlyblended mixture can then be shaped or loaded into tablets, capsules,pills, canisters, cartridges, dispensers and the like using conventionalprocedures and equipment.

In one embodiment, the pharmaceutical compositions of this invention aresuitable for inhaled administration. Suitable pharmaceuticalcompositions for inhaled administration will typically be in the form ofan aerosol or a powder. Such compositions are generally administeredusing well-known delivery devices, such as a nebulizer inhaler, ametered-dose inhaler (MDI), a dry powder inhaler (DPI) or a similardelivery device.

In a specific embodiment of this invention, the pharmaceuticalcomposition comprising the active agent is administered by inhalationusing a nebulizer inhaler. Such nebulizer devices typically produce astream of high velocity air that causes the pharmaceutical compositioncomprising the active agent to spray as a mist that is carried into thepatient's respiratory tract. Accordingly, when formulated for use in anebulizer inhaler, the active agent is typically dissolved in a suitablecarrier to form a solution. Alternatively, the active agent can bemicronized and combined with a suitable carrier to form a suspension ofmicronized particles of respirable size, where micronized is typicallydefined as having about 90% or more of the particles with a diameter ofless than about 10 μm. Suitable nebulizer devices are providedcommercially, for example, by PARI GmbH (Starnberg, German). Othernebulizer devices include Respimat (Boehringer Ingelheim) and thosedisclosed, for example, in U.S. Pat. No. 6,123,068 to Lloyd et al. andWO 97/12687 (Eicher et al.).

A representative pharmaceutical composition for use in a nebulizerinhaler comprises an isotonic aqueous solution comprising from about0.05 μg/mL to about 10 mg/mL of a compound of formula I or apharmaceutically acceptable salt or solvate or stereoisomer thereof.

In another specific embodiment of this invention, the pharmaceuticalcomposition comprising the active agent is administered by inhalationusing a dry powder inhaler. Such dry powder inhalers typicallyadminister the active agent as a free-flowing powder that is dispersedin a patient's air-stream during inspiration. In order to achieve a freeflowing powder, the active agent is typically formulated with a suitableexcipient such as lactose or starch.

A representative pharmaceutical composition for use in a dry powderinhaler comprises dry lactose having a particle size between about 1 μmand about 100 μm and micronized particles of a compound of formula I, ora pharmaceutically acceptable salt or solvate or stereoisomer thereof.

Such a dry powder formulation can be made, for example, by combining thelactose with the active agent and then dry blending the components.Alternatively, if desired, the active agent can be formulated without anexcipient. The pharmaceutical composition is then typically loaded intoa dry powder dispenser, or into inhalation cartridges or capsules foruse with a dry powder delivery device.

Examples of dry powder inhaler delivery devices include Diskhaler(GlaxoSmithKline, Research Triangle Park, N.C.) (see, e.g., U.S. Pat.No. 5,035,237 to Newell et al.); Diskus (GlaxoSmithKline) (see, e.g.,U.S. Pat. No. 6,378,519 to Davies et al.); Turbuhaler (AstraZeneca,Wilmington, Del.) (see, e.g., U.S. Pat. No. 4,524,769 to Wetterlin);Rotahaler (GlaxoSmithKtine) (see, e.g., U.S. Pat. No. 4,353,365 toHallworth et al.) and Handihaler (Boehringer Ingeiheim). Furtherexamples of suitable DPI devices are described in U.S. Pat. Nos.5,415,162 to Casper et al., 5,239,993 to Evans, and 5,715,810 toArmstrong et al., and references cited therein.

In yet another specific embodiment of this invention, the pharmaceuticalcomposition comprising the active agent is administered by inhalationusing a metered-dose inhaler. Such metered-dose inhalers typicallydischarge a measured amount of the active agent or a pharmaceuticallyacceptable salt or solvate or stereoisomer thereof using compressedpropellant gas. Accordingly, pharmaceutical compositions administeredusing a metered-dose inhaler typically comprise a solution or suspensionof the active agent in a liquefied propellant. Any suitable liquefiedpropellant may be employed including chlorofluorocarbons, such as CCl₃F,and hydrofluoroalkanes (HFAs), such as 1,1,1,2-tetrafluoroethane (HFA134a) and 1,1,1,2,3,3,3-heptafluoro-n-propane, (HFA 227). Due toconcerns about chlorofluorocarbons affecting the ozone layer,formulations containing HFAs are generally preferred. Additionaloptional components of HFA formulations include co-solvents, such asethanol or pentane, and surfactants, such as sorbitan trioleate, oleicacid, lecithin, and glycerin. See, for example, U.S. Pat. No. 5,225,183to Purewal et al., EP 0717987 A2 (Minnesota Mining and ManufacturingCompany), and WO 92/22286 (Minnesota Mining and Manufacturing Company).

A representative pharmaceutical composition for use in a metered-doseinhaler comprises from about 0.01% to about 5% by weight of a compoundof formula I, or a pharmaceutically acceptable salt or solvate orstereoisomer thereof; from about 0% to about 20% by weight ethanol; andfrom about 0% to about 5% by weight surfactant; with the remainder beingan HFA propellant.

Such compositions are typically prepared by adding chilled orpressurized hydrofluoroalkane to a suitable container containing theactive agent, ethanol (if present) and the surfactant (if present). Toprepare a suspension, the active agent is micronized and then combinedwith the propellant. The formulation is then loaded into an aerosolcanister, which forms a portion of a metered-dose inhaler device.Examples of metered-dose inhaler devices developed specifically for usewith HFA propellants are provided in U.S. Pat. Nos. 6,006,745 to Mareckiand 6,143,277 to Ashurst et al. Alternatively, a suspension formulationcan be prepared by spray drying a coating of surfactant on micronizedparticles of the active agent. See, for example, WO 99/53901 (GlaxoGroup Ltd.) and WO 00/61108 (Glaxo Group Ltd.).

For additional examples of processes of preparing respirable particles,and formulations and devices suitable for inhalation dosing see U.S.Pat. Nos. 6,268,533 to Gao et al., 5,983,956 to Trofast, 5,874,063 toBriggner et al., and 6,221,398 to Jakupovic et al.; and WO 99/55319(Glaxo Group Ltd.) and WO 00/30614 (AstraZeneca AB).

In another embodiment, the pharmaceutical compositions of this inventionare suitable for oral administration. Suitable pharmaceuticalcompositions for oral administration may be in the form of capsules,tablets, pills, lozenges, cachets, dragees, powders, granules; or as asolution or a suspension in an aqueous or non-aqueous liquid; or as anoil-in-water or water-in-oil liquid emulsion; or as an elixir or syrup;and the like; each containing a predetermined amount of a compound ofthe present invention as an active ingredient.

When intended for oral administration in a solid dosage form (i.e., ascapsules, tablets, pills and the like), the pharmaceutical compositionsof this invention will typically comprise a compound of the presentinvention as the active ingredient and one or more pharmaceuticallyacceptable carriers, such as sodium citrate or dicalcium phosphate.Optionally or alternatively, such solid dosage forms may also comprise:(1) fillers or extenders, such as starches, lactose, sucrose, glucose,mannitol, and/or silicic acid; (2) binders, such ascarboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,sucrose and/or acacia; (3) humectants, such as glycerol; (4)disintegrating agents, such as agar-agar, calcium carbonate, potato ortapioca starch, alginic acid, certain silicates, and/or sodiumcarbonate; (5) solution retarding agents, such as paraffin; (6)absorption accelerators, such as quaternary ammonium compounds; (7)wetting agents, such as cetyl alcohol and/or glycerol monostearate; (8)absorbents, such as kaolin and/or bentonite clay; (9) lubricants, suchas talc, calcium stearate, magnesium stearate, solid polyethyleneglycols, sodium lauryl sulfate, and/or mixtures thereof; (10) coloringagents; and (11) buffering agents.

Release agents, wetting agents, coating agents, sweetening, flavoringand perfuming agents, preservatives and antioxidants can also be presentin the pharmaceutical compositions of this invention. Examples ofpharmaceutically acceptable antioxidants include: (1) water-solubleantioxidants, such as ascorbic acid, cysteine hydrochloride, sodiumbisulfate, sodium metabisulfate sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal-chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like. Coating agents fortablets, capsules, pills and like, include those used for entericcoatings, such as cellulose acetate phthalate (CAP), polyvinyl acetatephthalate (PVAP), hydroxypropyl methylcellulose phthalate, methacrylicacid-methacrylic acid ester copolymers, cellulose acetate trimellitate(CAT), carboxymethyl ethyl cellulose (CMEC), hydroxypropyl methylcellulose acetate succinate (HPMCAS), and the like.

If desired, the pharmaceutical compositions of the present invention mayalso be formulated to provide slow or controlled release of the activeingredient using, by way of example, hydroxypropyl methyl cellulose invarying proportions; or other polymer matrices, liposomes and/ormicrospheres.

In addition, the pharmaceutical compositions of the present inventionmay optionally contain opacifying agents and may be formulated so thatthey release the active ingredient only, or preferentially, in a certainportion of the gastrointestinal tract, optionally, in a delayed manner.Examples of embedding compositions which can be used include polymericsubstances and waxes. The active ingredient can also be inmicro-encapsulated form, if appropriate, with one or more of theabove-described excipients.

Suitable liquid dosage forms for oral administration include, by way ofillustration, pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. Such liquid dosage formstypically comprise the active ingredient and an inert diluent, such as,for example, water or other solvents, solubilizing agents andemulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate,ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol,1,3-butylene glycol, oils (e.g., cottonseed, groundnut, corn, germ,olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol,polyethylene glycols and fatty acid esters of sorbitan, and mixturesthereof. Suspensions, in addition to the active ingredient, may containsuspending agents such as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminium metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

When intended for oral administration, the pharmaceutical compositionsof this invention are preferably packaged in a unit dosage form. Theterm “unit dosage form” means a physically discrete unit suitable fordosing a patient, i.e., each unit containing a predetermined quantity ofactive agent calculated to produce the desired therapeutic effect eitheralone or in combination with one or more additional units. For example,such unit dosage forms may be capsules, tablets, pills, and the like.

The compounds of this invention can also be administered transdermallyusing known transdermal delivery systems and excipients. For example, acompound of this invention can be admixed with permeation enhancers,such as propylene glycol, polyethylene glycol monolaurate,azacycloalkan-2-ones and the like, and incorporated into a patch orsimilar delivery system. Additional excipients including gelling agents,emulsifiers and buffers, may be used in such transdermal compositions ifdesired.

The pharmaceutical compositions of this invention may also contain othertherapeutic agents that are co-administered with a compound of formulaI, or pharmaceutically acceptable salt or solvate or stereoisomerthereof. For example, the pharmaceutical compositions of this inventionmay further comprise one or more therapeutic agents selected from otherbronchodilators (e.g., PDE₃ inhibitors, adenosine 2b modulators and β₂adrenergic receptor agonists); anti-inflammatory agents (e.g., steroidalanti-inflammatory agents, such as corticosteroids; non-steroidalanti-inflammatory agents (NSAIDs), and PDE₄ inhibitors); othermuscarinic receptor antagonists (i.e., anticholinergic agents);antiinfective agents (e.g., Gram positive and Gram negative antibioticsor antivirals); antihistamines; protease inhibitors; and afferentblockers (e.g., D₂ agonists and neurokinin modulators). In oneparticular aspect of the invention, the compound of the invention isco-administered with a β₂ adrenergic receptor agonist and a steroidalanti-inflammatory agent. The other therapeutic agents can be used in theform of pharmaceutically acceptable salts or solvates. Additionally, ifappropriate, the other therapeutic agents can be used as optically purestereoisomers.

Representative β₂ adrenergic receptor agonists that can be used incombination with the compounds of this invention include, but are notlimited to, salmeterol, salbutamol, formoterol, salmefamol, fenoterol,terbutaline, albuterol, isoetharine, metaproterenol, bitolterol,pirbuterol, levalbuterol and the like, or pharmaceutically acceptablesalts thereof. Other β₂ adrenergic receptor agonists that can be used incombination with the compounds of this invention include, but are notlimited to,3-(4-{[6-({(2R)-2-hydroxy-2-[4-hydroxy-3-(hydroxymethyl)-phenyl]ethyl}amino)-hexyl]oxy}butyl)benzenesulfonamideand3-(-3-{[7({(2R)-2-hydroxy-2-[4-hydroxy-3-(hydroxymethyl)phenyl]ethyl}-amino)heptyl]oxy}-propyl)benzenesulfonamideand related compounds disclosed in WO 02/066422 (Glaxo Group Ltd.);3-[3-(4-{[6-([(2R)-2-hydroxy-2-[4-hydroxy-3-(hydroxymethyl)phenyl]ethyl}amino)hexyl]oxy}butyl)-phenyl]imidazolidine-2,4-dioneand related compounds disclosed in WO 02/070490 (Glaxo Group Ltd.);3-(4-{[6-({(2R)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)hexyl]oxy}butyl)-benzenesulfonamide,3-(4-{[6-({(2S)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)hexyl]oxy}butyl)-benzenesulfonamide,3-(4-{[6-({(2R/S)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)hexyl]oxy}butyl)-benzenesulfonamide,N-(tert-butyl)-3-(4-{[6-({(2R)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)hexyl]-oxy}butyl)benzenesulfonamide,N-(tert-butyl)-3-(4-{([6-({(2S)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)-hexyl]oxy}butyl)-benzenesulfonamide,N-(tert-butyl)-3-(4-{[6-({(2R/S)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)hexyl]-oxy}butyl)benzenesulfonamide and related compounds disclosed inWO 02/076933 (Glaxo Group Ltd.);4-{(1R)-2-[(6-{2-[(2,6-dichlorobenzyl)oxy]ethoxy}hexyl)amino]-1-hydroxyethyl}-2-(hydroxymethyl)phenol and relatedcompounds disclosed in WO 03/024439 (Glaxo Group Ltd.);N-{2-[4-((R)-2-hydroxy-2-phenylethylamino)phenyl]ethyl}-(R)-2-hydroxy-2-(3-formamido-4-hydroxyphenyl)ethylamineand related compounds disclosed in U.S. Pat. No. 6,576,793 to Moran etal.;N-{2-[4(3-phenyl-4-methoxyphenyl)aminophenyl]ethyl}-(R)-2-hydroxy-2-(8-hydroxy-2(1H)-quinolinon-5-yl)ethylamineand related compounds disclosed in U.S. Pat. No. 6,653,323 to Moran etal.; and pharmaceutically acceptable salts thereof. In a particularembodiment, the β₂-adrenoreceptor agonist is a crystallinemonohydrochloride salt ofN-{2-[4-((R)-2-hydroxy-2-phenylethylamino)phenyl]ethyl}-(R)-2-hydroxy-2-(3-formamido-4-hydroxyphenyl)ethylamine. When employed, the β₂-adrenoreceptor agonist will be presentin the pharmaceutical composition in a therapeutically effective amount.Typically, the β₂-adrenoreceptor agonist will be present in an amountsufficient to provide from about 0.05 μg to about 500 μg per dose.

Representative steroidal anti-inflammatory agents that can be used incombination with the compounds of this invention include, but are notlimited to, methyl prednisolone, prednisolone, dexamethasone,fluticasone propionate,6α,9α-difluoro-17β-[(2-furanylcarbonyl)oxy]-11β-hydroxy-16α-methyl-3-oxoandrosta-1,4-diene-17α-carbothioicacid S-fluoromethyl ester,6α,9α-difluoro-11β-hydroxy-16α-methyl-3-oxo-17α-propionyloxy-androsta-1,4-diene-17β-carbothioicacid S-(2-oxo-tetrahydrofuran-3S-yl) ester, beclomethasone esters (e.g.,the 17-propionate ester or the 17,21-dipropionate ester), budesonide,flunisolide, mometasone esters (e.g., the furoate ester), triamcinoloneacetonide, rofleponide, ciclesonide, butixocort propionate, RPR-106541,ST-1126 and the like, or pharmaceutically-acceptable salts thereof. Whenemployed, the steroidal anti-inflammatory agent will be present in thepharmaceutical composition in a therapeutically effective amount.Typically, the steroidal anti-inflammatory agent will be present in anamount sufficient to provide from about 0.05 μg to about 500 μg perdose.

An exemplary combination is a compound of formula I, or pharmaceuticallyacceptable salt or solvate or stereoisomer thereof, co-administered withsalmeterol as the β₂ adrenergic receptor agonist, and fluticasonepropionate as the steroidal anti-inflammatory agent. Another exemplarycombination is a compound of formula I, or pharmaceutically acceptablesalt or solvate or stereoisomer thereof, co-administered with acrystalline monohydrochloride salt ofN-{2-[4-((R)-2-hydroxy-2-phenylethylamino)phenyl]ethyl}-(R)-2-hydroxy-2-(3-formamido-4-hydroxyphenyl)ethylamineas the β₂-adrenoreceptor agonist, and6α,9α-difluoro-17α-[(2-furanylcarbonyl)oxy]-11β-hydroxy-16α-methyl-3-oxoandrosta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester as the steroidal anti-inflammatory agent.

Other suitable combinations include, for example, otheranti-inflammatory agents, e.g., NSAIDs (such as sodium cromoglycate;nedocromil sodium; phosphodiesterase (PDE) inhibitors (e.g.,theophylline, PDE4 inhibitors or mixed PDE3/PDE4 inhibitors);leukotriene antagonists (e.g., monteleukast); inhibitors of leukotrienesynthesis; iNOS inhibitors; protease inhibitors, such as tryptase andelastase inhibitors; beta-2 integrin antagonists and adenosine receptoragonists or antagonists (e.g., adenosine 2a agonists); cytokineantagonists (e.g., chemokine antagonists such as, an interleukinantibody (αIL antibody), specifically, an αIL-4 therapy, an αIL-13therapy, or a combination thereof); or inhibitors of cytokine synthesis.

For example, representative phosphodiesterase-4 (PDE4) inhibitors ormixed PDE3/PDE4 inhibitors that can be used in combination with thecompounds of this invention include, but are not limited to cis4-cyano-4-(3-cyclopentyloxy-4-methoxyphenyl)cyclohexan-1-carboxylicacid,2-carbomethoxy-4-cyano-4-(3-cyclopropylmethoxy-4-difluoromethoxyphenyl)cyclohexan-1-one;cis-[4-cyano-4-(3-cyclopropylmethoxy-4-difluoromethoxyphenyl)cyclohexan-1-ol];cis-4-cyano-4-[3-(cyclopentyloxy)-4-methoxyphenyl]cyclohexane-1-carboxylicacid and the like, or pharmaceutically acceptable salts thereof. Otherrepresentative PDE4 or mixed PDE4/PDE3 inhibitors include AWD-12-281(elbion); NCS-613 (INSERM); D-4418 (Chiroscience and Schering-Plough);CI-1018 or PD-168787 (Pfizer); benzodioxole compounds disclosed inWO99/16766 (Kyowa Hakko); K-34 (Kyowa Hakko); V-11294A (Napp);roflumilast (Byk-Gulden); phthalazinone compounds disclosed inWO99/47505 (Byk-Gulden); Pumafentrine (Byk-Gulden, now Altana);arofylline (Almirall-Prodesfarma); VM554/UM565 (Vernalis); T-440 (TanabeSeiyaku); and T2585 (Tanabe Seiyaku).

Representative muscarinic antagonists (i.e., anticholinergic agents)that can be used in combination with, and in addition to, the compoundsof this invention include, but are not limited to, atropine, atropinesulfate, atropine oxide, methylatropine nitrate, homatropinehydrobromide, hyoscyamine (d, l) hydrobromide, scopolamine hydrobromide,ipratropium bromide, oxitropium bromide, tiotropium bromide,methantheline, propantheline bromide, anisotropine methyl bromide,clidinium bromide, copyrrolate (Robinul), isopropamide iodide,mepenzolate bromide, tridihexethyl chloride (Pathilone), hexocycliummethylsulfate, cyclopentolate hydrochloride, tropicamide,trihexyphenidyl hydrochloride, pirenzepine, telenzepine, AF-DX 116 andmethoctramine and the like, or a pharmaceutically acceptable saltthereof, or, for those compounds listed as a salt, alternatepharmaceutically acceptable salt thereof.

Representative antihistamines (i.e., H₁-receptor antagonists) that canbe used in combination with the compounds of this invention include, butare not limited to, ethanolamines, such as carbinoxamine maleate,clemastine fumarate, diphenylhydramine hydrochloride and dimenhydrinate;ethylenediamines, such as pyrilamine amleate, tripelennaminehydrochloride and tripelennamine citrate; alkylamines, such aschlorpheniramine and acrivastine; piperazines, such as hydroxyzinehydrochloride, hydroxyzine pamoate, cyclizine hydrochloride, cyclizinelactate, meclizine hydrochloride and cetirizine hydrochloride;piperidines, such as astemizole, levocabastine hydrochloride, loratadineor its descarboethoxy analogue, terfenadine and fexofenadinehydrochloride; azelastine hydrochloride; and the like, or apharmaceutically acceptable salt thereof; or, for those compounds listedas a salt, alternate pharmaceutically acceptable salt thereof.

Suitable doses for the other therapeutic agents administered incombination with a compound of the invention are in the range of about0.05 μg/day to about 100 mg/day.

The following formulations illustrate representative pharmaceuticalcompositions of the present invention:

FORMULATION EXAMPLE A

A dry powder for administration by inhalation is prepared as follows:Ingredients Amount Compound of the invention 0.2 mg Lactose  25 mg

Representative Procedure: The compound of the invention is micronizedand then blended with lactose. This blended mixture is then loaded intoa gelatin inhalation cartridge. The contents of the cartridge areadministered using a powder inhaler.

FORMULATION EXAMPLE B

A dry powder formulation for use in a dry powder inhalation device isprepared as follows:

Representative Procedure: A pharmaceutical composition is preparedhaving a bulk formulation ratio of micronized compound of the inventionto lactose of 1:200. The composition is packed into a dry powderinhalation device capable of delivering between about 10 μg and about100 μg of the compound of the invention per dose.

FORMULATION EXAMPLE C

A dry powder for administration by inhalation in a metered dose inhaleris prepared as follows:

Representative Procedure: A suspension containing 5 wt % of a compoundof the invention and 0.1 wt % lecithin is prepared by dispersing 10 g ofthe compound of the invention as micronized particles with mean sizeless than 10 μm in a solution formed from 0.2 g of lecithin dissolved in200 mL of demineralized water. The suspension is spray dried and theresulting material is micronized to particles having a mean diameterless than 1.5 μm. The particles are loaded into cartridges withpressurized 1,1,1,2-tetrafluoroethane.

FORMULATION EXAMPLE D

A pharmaceutical composition for use in a metered dose inhaler isprepared as follows:

Representative Procedure: A suspension containing 5 wt % compound of theinvention, 0.5 wt % lecithin, and 0.5 wt % trehalose is prepared bydispersing 5 g of active ingredient as micronized particles with meansize less than 10 μm in a colloidal solution formed from 0.5 g oftrehalose and 0.5 g of lecithin dissolved in 100 mL of demineralizedwater. The suspension is spray dried and the resulting material ismicronized to particles having a mean diameter less than 1.5 μm. Theparticles are loaded into canisters with pressurized1,1,1,2-tetrafluoroethane.

FORMULATION EXAMPLE E

A pharmaceutical composition for use in a nebulizer inhaler is preparedas follows:

Representative Procedure: An aqueous aerosol formulation for use in anebulizer is prepared by dissolving 0.1 mg of the compound of theinvention in 1 mL of a 0.9% sodium chloride solution acidified withcitric acid. The mixture is stirred and sonicated until the activeingredient is dissolved. The pH of the solution is adjusted to a valuein the range of from 3 to 8 by the slow addition of NaOH.

FORMULATION EXAMPLE F

Hard gelatin capsules for oral administration are prepared as follows:Ingredients Amount Compound of the invention 250 mg Lactose(spray-dried) 200 mg Magnesium stearate  10 mg

Representative Procedure: The ingredients are thoroughly blended andthen loaded into a hard gelatin capsule (460 mg of composition percapsule).

FORMULATION EXAMPLE G

A suspension for oral administration is prepared as follows: IngredientsAmount Compound of the invention 1.0 g Fumaric acid 0.5 g Sodiumchloride 2.0 g Methyl paraben 0.15 g Propyl paraben 0.05 g Granulatedsugar 25.5 g Sorbitol (70% solution) 12.85 g Veegum k (Vanderbilt Co.)1.0 g Flavoring 0.035 mL Colorings 0.5 mg Distilled water q.s. to 100 mL

Representative Procedure: The ingredients are mixed to form a suspensioncontaining 100 mg of active ingredient per 10 mL of suspension.

FORMULATION EXAMPLE H

An injectable formulation is prepared as follows: Ingredients AmountCompound of the invention 0.2 g Sodium acetate buffer solution (0.4 M)2.0 mL HCl (0.5 N) or NaOH (0.5 N) q.s. to pH 4 Water (distilled,sterile) q.s. to 20 mL

Representative Procedure: The above ingredients are blended and the pHis adjusted to 4±0.5 using 0.5 N HCl or 0.5 N NaOH.

Utility

The biphenyl compounds of this invention are expected to be useful asmuscarinic receptor antagonists and therefore, such compounds areexpected to be useful for treating medical conditions mediated bymuscarinic receptors, i.e., medical conditions which are ameliorated bytreatment with a muscarinic receptor antagonist. Such medical conditionsinclude, by way of example, pulmonary disorders or diseases includingthose associated with reversible airway obstruction, such as chronicobstructive pulmonary disease (e.g., chronic and wheezy bronchitis andemphysema), asthma, pulmonary fibrosis, allergic rhinitis, rhinorrhea,and the like. Other medical conditions that can be treated withmuscarinic receptor antagonists are genitourinary tract disorders, suchas overactive bladder or detrusor hyperactivity and their symptoms;gastrointestinal tract disorders, such as irritable bowel syndrome,diverticular disease, achalasia, gastrointestinal hypermotilitydisorders and diarrhea; cardiac arrhythmias, such as sinus bradycardia;Parkinson's disease; cognitive disorders, such as Alzheimer's disease;dismenorrhea; and the like.

In one embodiment, the compounds of this invention are useful fortreating smooth muscle disorders in mammals, including humans and theircompanion animals (e.g., dogs, cats etc.). Such smooth muscle disordersinclude, by way of illustration, overactive bladder, chronic obstructivepulmonary disease and irritable bowel syndrome.

When used to treat smooth muscle disorders or other conditions mediatedby muscarinic receptors, the compounds of this invention will typicallybe administered orally, rectally, parenterally or by inhalation in asingle daily dose or in multiple doses per day. The amount of activeagent administered per dose or the total amount administered per daywill typically be determined by the patient's physician and will dependon such factors as the nature and severity of the patients condition,the condition being treated, the age and general health of the patient,the tolerance of the patient to the active agent, the route ofadministration and the like.

Typically, suitable doses for treating smooth muscle disorders or otherdisorders mediated by muscarinic receptors will range from about 0.14μg/kg/day to about 7 mg/kg/day of active agent; including from about0.15 μg/kg/day to about 5 mg/kg/day. For an average 70 kg human, thiswould amount to about 10 μg per day to about 500 mg per day of activeagent.

In a specific embodiment, the compounds of this invention are useful fortreating pulmonary or respiratory disorders, such as COPD or asthma, inmammals including humans. When used to treat such disorders, thecompounds of this invention will typically be administered by inhalationin multiple doses per day, in a single daily dose or a single weeklydose. Generally, the dose for treating a pulmonary disorder will rangefrom about 10 μg/day to about 200 μg/day. As used herein, COPD includeschronic obstructive bronchitis and emphysema (see, for example, Barnes,Chronic Obstructive Pulmonary Disease, N Engl J Med 343:269-78 (2000)).

When used to treat a pulmonary disorder, the compounds of this inventionare optionally administered in combination with other therapeutic agentssuch as a β₂-adrenoreceptor agonist; a corticosteroid, a non-steroidalanti-inflammatory agent, or combinations thereof.

When administered by inhalation, the compounds of this inventiontypically have the effect of producing bronchodilation. Accordingly, inanother of its method aspects, this invention is directed to a method ofproducing bronchodilation in a patient, the method comprisingadministering to a patient a bronchodilation-producing amount of acompound of the invention. Generally, the therapeutically effective dosefor producing bronchodilation will range from about 10 μg/day to about200 μg/day.

In another embodiment, the compounds of this invention are used to treatoveractive bladder. When used to treat overactive bladder, the compoundsof this invention will typically be administered orally in a singledaily dose or in multiple doses per day; preferably in a single dailydose. Preferably, the dose for treating overactive bladder will rangefrom about 1.0 to about 500 mg/day.

In yet another embodiment, the compounds of this invention are used totreat irritable bowel syndrome. When used to treat irritable bowelsyndrome, the compounds of this invention will typically be administeredorally or rectally in a single daily dose or in multiple doses per day.Preferably, the dose for treating irritable bowel syndrome will rangefrom about 1.0 to about 500 mg/day.

Since compounds of this invention are muscarinic receptor antagonists,such compounds are also useful as research tools for investigating orstudying biological systems or samples having muscarinic receptors. Suchbiological systems or samples may comprise M₁, M₂, M₃, M₄ and/or M₅muscarinic receptors. Any suitable biological system or sample havingmuscarinic receptors may be employed in such studies which may beconducted either in vitro or in vivo. Representative biological systemsor samples suitable for such studies include, but are not limited to,cells, cellular extracts, plasma membranes, tissue samples, mammals(such as mice, rats, guinea pigs, rabbits, dogs, pigs, etc.), and thelike.

In this embodiment, a biological system or sample comprising amuscarinic receptor is contacted with a muscarinic receptor-antagonizingamount of a compound of this invention. The effects of antagonizing themuscarinic receptor are then determined using conventional proceduresand equipment, such as radioligand binding assays and functional assays.Such functional assays include ligand-mediated changes in intracellularcyclic adenosine monophosphate (cAMP), ligand-mediated changes inactivity of the enzyme adenylyl cyclase (which synthesizes cAMP),ligand-mediated changes in incorporation of guanosine5′-O-(γ-thio)triphosphate ([³⁵S]GTPγS) into isolated membranes viareceptor catalyzed exchange of [³⁵S]GTPγS for GDP, ligand-mediatedchanges in free intracellular calcium ions (measured, for example, witha fluorescence-linked imaging plate reader or FLIPR® from MolecularDevices, Inc.). A compound of this invention will antagonize or decreasethe activation of muscarinic receptors in any of the functional assayslisted above, or assays of a similar nature. A muscarinicreceptor-antagonizing amount of a compound of this invention willtypically range from about 0.1 nanomolar to about 100 nanomolar.

Additionally, the compounds of this invention can be used as researchtools for discovering new compounds that have muscarinic receptorantagonist activity. In this embodiment, muscarinic receptor bindingdata (e.g., as determined by in vitro radioligand displacement assays)for a test compound or a group of test compounds is compared to themuscarinic receptor binding data for a compound of this invention toidentify those test compounds that have about equal or superiormuscarinic receptor binding, if any. This aspect of the inventionincludes, as separate embodiments, both the generation of comparisondata (using the appropriate assays) and the analysis of the test data toidentify test compounds of interest.

In another embodiment, the compounds of this invention are used toantagonize a muscarinic receptor in biological system, and a mammal inparticular, such as mice, rats, guinea pigs, rabbits, dogs, pigs, humansand so forth. In this embodiment, a therapeutically effective amount ofthe compound of formula I is administered to the mammal. The effects ofantagonizing the muscarinic receptor can then determined usingconventional procedures and equipment, examples of which are describedabove.

Among other properties, compounds of this invention have been found tobe potent inhibitors of M₃ muscarinic receptor activity. Accordingly, ina specific embodiment, this invention is directed to compounds offormula I having an inhibition dissociation constant (K_(i)) for the M₃receptor subtype of less than or equal to 10 nM; preferably, less thanor equal to 5 nM; (as determined, for example, by an in vitroradioligand displacement assay).

Additionally, compounds of this invention have also been found topossess surprising and unexpected duration of action. Accordingly, inanother specific embodiment, this invention is directed to compounds offormula I having a duration of action greater than or equal to about 24hours.

Moreover, compounds of this invention have been found to possess reducedside effects, such as dry mouth, at efficacious doses when administeredby inhalation compared to other known muscarinic receptor antagonistsadministered by inhalation (such as tiotropium).

These properties, as well as the utility of the compounds of thisinvention, can be demonstrated using various in vitro and in vivo assayswell-known to those skilled in the art. For example, representativeassays are described in further detail in the following Examples.

EXAMPLES

The following Preparations and Examples illustrate specific embodimentsof this invention. In these examples, the following abbreviations havethe following meanings:

-   -   AC adenylyl cyclase    -   ACh acetylcholine    -   ACN acetonitrile    -   BSA bovine serum albumin    -   cAMP 3′-5′ cyclic adenosine monophosphate    -   CHO Chinese hamster ovary    -   cM₅ cloned chimpanzee M₅ receptor    -   DCM dichloromethane (i.e., methylene chloride)    -   DIBAL diisobutylaluminium hydride    -   DIPEA N,N-diisopropylethylamine    -   dPBS Dulbecco's phosphate buffered saline    -   DMF dimethylformamide    -   DMSO dimethyl sulfoxide    -   EDC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide    -   EDCI 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride    -   EDTA ethylenediaminetetraacetic acid    -   EtOAc ethyl acetate    -   EtOH ethanol    -   FBS fetal bovine serum    -   FLIPR fluorometric imaging plate reader    -   HATU O-(7-azabenzotriazol-1-yl-N,N,N′,N′-tetramethyluronium        hexafluorophosphate    -   HBSS Hank's buffered salt solution    -   HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid    -   HOAt 1-hydroxy-7-azabenzotriazole    -   hM₁ cloned human M₁ receptor    -   hM₂ cloned human M₂ receptor    -   hM₃ cloned human M₃ receptor    -   hM₄ cloned human M₄ receptor    -   hM₅ cloned human M₅ receptor    -   HOBT 1-hydroxybenzotriazole hydrate    -   HPLC high-performance liquid chromatography    -   IPA isopropanol    -   MCh methylcholine    -   MTBE methyl t-butyl ether    -   TFA trifluoroacetic acid    -   THF tetrahydrofuran

Any other abbreviations used herein but not defined have their standard,generally accepted meaning. Unless noted otherwise, all materials, suchas reagents, starting materials and solvents, were purchased fromcommercial suppliers (such as Sigma-Aldrich, Fluka, and the like) andwere used without further purification.

Unless otherwise indicated, HPLC analysis was conducted using an Agilent(Palo Alto, Calif.) Series 1100 instrument equipped with a Zorbax BonusRP 2.1×50 mm column (Agilent) having a 3.5 micron particle size.Detection was by UV absorbance at 214 nm. The mobile phases employedwere as follows (by volume): A is ACN (2%), water (98%) and TFA. (0.1%);and B is ACN (90%), water (10%) and TFA (0.1%). HPLC 10-70 data wasobtained using a flow rate of 0.5 mL/minute of 10 to 70% B over a 6minute gradient (with the remainder being A). Similarly, HPLC 5-35 dataand HPLC 10-90 data were obtained using 5 to 35% B; or 10 to 90% B overa 5 minute gradient.

Liquid chromatography mass spectrometry (LCMS) data were obtained withan Applied Biosystems (Foster City, Calif.) Model API-150EX instrument.LCMS 10-90 data was obtained using 10 to 90% Mobile Phase B over a 5minute gradient.

Small-scale purification was conducted using an API 150EX PrepWorkstation system from Applied Biosystems. The mobile phases employedwere as follows (by volume): A is water and 0.05% TFA; and B is ACN and0.05% TFA. For arrays (typically about 3 to 50 mg recovered sample size)the following conditions were used: 20 mL/min flow rate; 15 minutegradients and a 20 mm×50 mm Prism RP column with 5 micron particles(Thermo Hypersil-Keystone, Bellefonte, Pa.). For larger scalepurifications (typically greater than 100 mg crude sample), thefollowing conditions were used: 60 mL/min flow rate; 30 minute gradientsand a 41.4 mm×250 mm Microsorb BDS column with 10 micron particles(Varian, Palo Alto, Calif.).

Preparation 1 Biphenyl-2-ylcarbamic Acid Piperidinyl Ester

Biphenyl-2-isocyanate (97.5 g, 521 mmol) and4-hydroxy-N-benzylpiperidine (105 g, 549 mmol) were heated together at70° C. for 12 hours. The reaction mixture was then cooled to 50° C. andEtOH (1 L) was added and then 6M HCl (191 mL) was added slowly. Theresulting mixture was then cooled to ambient temperature and ammoniumformate (98.5 g, 1.56 mol) was added and then nitrogen gas was bubbledthrough the solution vigorously for 20 minutes. Palladium on activatedcarbon (20 g, 10 wt. % dry basis) was then added and the reactionmixture was heated at 40° C. for 12 hours, and then filtered through apad of Celite. The solvent was then removed under reduced pressure and1M HCl (40 mL) was added to the crude residue. The pH of the mixture wasthen adjusted with 10 N NaOH to pH 12. The aqueous layer was extractedwith ethyl acetate (2×150 mL) and the organic layer was dried (magnesiumsulfate), filtered and the solvent removed under reduced pressure togive 155 g of the title intermediate (100% yield). HPLC (10-70)R_(t)=2.52; m/z: [M+H⁺] calcd for C₁₈H₂₀N₂O₂, 297.15; found, 297.3.

Preparation 2 N-Benzyl-N-methylaminoacetaldehyde

To a 3-necked 2-L flask was added N-benzyl-N-methylethanolamine (30.5 g,0.182 mol), DCM (0.5 L), DIPEA (95 mL, 0.546 mol) and DMSO (41 mL, 0.728mol). Using an ice bath, the mixture was cooled to about −10° C. andsulfur trioxide pyridine-complex (87 g, 0.546 mol) was added in 4portions over 5 minute intervals. The reaction was stirred at −10° C.for 2 hours. Before removing the ice-bath, the reaction was quenched byadding water (0.5 L). The aqueous layer was separated and the organiclayer was washed with water (0.5 L) and brine (0.5 L) and then driedover magnesium sulfate and filtered to provide the title compound whichwas used without further purification.

Preparation 3 Biphenyl-2-ylcarbamic Acid1-[2-(Benzylmethylamino)ethyl]piperidin-4-yl Ester

To a 2-L flask, containing the product of Preparation 2 in DCM (0.5 L)was added the product of Preparation 1 (30 g, 0.101 mol) followed bysodium triacetoxyborohydride (45 g, 0.202 mol). The reaction mixture wasstirred overnight and then quenched by the addition of 1 N hydrochloricacid (0.5 L) with vigorous stirring. Three layers were observed and theaqueous layer was removed. After washing with 1N NaOH (0.5 L), ahomogenous organic layer was obtained which was then washed with asaturated solution of aqueous NaCl (0.5 L), dried over magnesiumsulfate, filtered and the solvent removed under reduced pressure. Theresidue was purified by dissolving it in a minimal amount of EPA andcooling this solution to 0° C. to form a solid which was collected andwashed with cool IPA to provide 42.6 g of the title compound (95%yield). MS m/z: [M+H⁺] calcd f for C₂₈H₃₃N₃O₂, 444.3; found, 444.6.R_(f)=3.51 min (10-70 ACN: H₂O, reverse phase HPLC).

Preparation 4 Biphenyl-2-ylcarbamic Acid1-(2-Methylaminoethyl)piperidin-4-yl Ester

To a Parr hydrogenation flask was added the product of Preparation 3 (40g, 0.09 mol) and EtOH (0.5 L). The flask was flushed with nitrogen gasand palladium on activated carbon (15 g, 10 wt. % (dry basis), 37%wt/wt) was added along with acetic acid (20 mL). The mixture was kept onthe Parr hydrogenator under a hydrogen atmosphere (˜50 psi) for 3 h. Themixture was then filtered and washed with EtOH. The filtrate wascondensed and the residue was dissolved in a minimal amount of DCM.Isopropyl acetate (10 volumes) was added slowly to form a solid whichwas collected to provide 22.0 g of the title compound (70% yield). MSm/z: [M+H⁺] calcd for C₂₁H₂₇N₃O₂, 354.2; found, 354.3. R_(f)=2.96 min(10-70 ACN: H₂O, reverse phase HPLC).

Preparation 5 Biphenyl-2-ylcarbamic Acid1-{2-[(4-Formylbenzoyl)methylamino]ethyl}piperidin-4-yl Ester

To a three-necked 1-L flask was added 4-carboxybenzaldehyde (4.77 g,31.8 mmol), EDC (6.64 g, 34.7 mmol), HOBT (1.91 g, 31.8 mmol), and DCM(200 mL). When the mixture was homogenous, a solution of the product ofPreparation 4 (10 g, 31.8 mmol) in DCM (100 mL) was added slowly. Thereaction mixture was stirred at room temperature for 16 hours and thenwashed with water (1×100 mL), 1N HCl (5×60 mL), 1N NaOH (1×100 mL) brine(1×50 mL), dried over sodium sulfate, filtered and concentrated toafford 12.6 g of the title compound (92% yield; 85% purity based onHPLC). MS m/z: [M+H⁺] calcd for C₂₉H₃₁N₃O₄, 486.2; found, 486.4.R^(f)3.12 min (10-70 ACN: H₂O, reverse phase HPLC).

Example 1 Biphenyl-2-ylcarbamic Acid1-(2-{[4(4-Carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylEster

To a three-necked 2-L flask was added isonipecotamide (5.99 g, 40.0mmol), acetic acid (2.57 mL), sodium sulfate (6.44 g) and IPA (400 mL).The reaction mixture was cooled to 0-10° C. with an ice bath and asolution of the product of Preparation 5 (11 g, 22.7 mmol) in IPA (300mL) was slowly added. The reaction mixture was stirred at roomtemperature for 2 hours and then cooled to 0-10° C. Sodiumtriacetoxyborohydride (15.16 g, 68.5 mmol) was added portion wise andthis mixture was stirred at room temperature for 16 h. The reactionmixture was then concentrated under reduced pressure to a volume ofabout 50 mL and this mixture was acidified with 1N HCl (200 mL) to pH 3.The resulting mixture was stirred at room temperature for 1 hour andthen extracted with DCM (3×250 mL). The aqueous phase was then cooled to0-5° C. with an ice bath and 50% aqueous NaOH solution was added toadjust the pH of the mixture to 10. This mixture was then extracted withisopropyl acetate (3×300 mL) and the combined organic layers were washedwith water (100 mL), brine (2×50 mL), dried over sodium sulfate,filtered and concentrated to afford 10.8 g of the title compound (80%yield. MS m/z: [M+H+] calcd for C₃₅H₄₃N₅O₄, 598.3; found, 598.6.R_(f)=2.32 min (10-70 ACN: H₂O, reverse phase HPLC).

Example 1A

Biphenyl-2-ylcarbamic acid1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylester was also prepared as a diphosphate salt using the followingprocedure:

5.0 g of the product of Example 1 was combined with 80 ml of IPA:ACN(1:1). 4.0 ml of water was added and the mixture heated to 50° C. understirring, forming a clear solution. To this was added dropwise at 50°C., 16 ml 1M phosphoric acid. The resulting cloudy solution was stirredat 50° C. for 5 hours, then allowed to cool to ambient temperature,under slow stirring, overnight. The resulting crystals were collected byfiltration and air-dried for 1 hour, then under vacuum for 18 hours, togive the diphosphate salt of the title compound (5.8 g, 75% yield) as awhite crystalline solid (98.3% purity by HPLC).

Example 1B

Biphenyl-2-ylcarbamic acid1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylester was also prepared as a monosulfate salt using the followingprocedure.

442 mg of the product of Example 1 (0.739 mmol of 96% pure material) wastaken up in 5 ml of H₂O:ACN (1:1) and 1.45 ml of IN sulfuric acid wasadded slowly, while monitoring the pH. The pH was adjusted to approx. pH3.3. The clear solution was filtered through a 0.2 micron filter, frozenand lyophilized to dryness. 161 g of the lyophilized material wasdissolved in 8.77 ml of IPA:ACN (10:1). The suspension was heated byplacing the vial in a pre-heated 70° C. water bath for 1.5 hours. Oildroplets formed within 5 minutes. The heat was lowered to 60° C. and themixture heated for an additional 1.5 hours, followed by heating at 50°C. for 40 minutes, at 40° C. for 40 minutes, then at 30° C. for 45minutes. The heat was turned off and the mixture was allowed to slowlycool to room temperature. The next day, the material was viewed under amicroscope and indicated needles and plates. The material was thenheated at 40° C. for 2 hours, at 35° C. for 30 minutes, and then at 30°C. for 30 minutes. The heat was turned off and the mixture was allowedto slowly cool to room temperature. The solid was then filtered anddried using a vacuum pump for 1 hour to give the monosulfate salt of thetitle compound (117 mg, 73% yield).

Example 1C

Biphenyl-2-ylcarbamic acid1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylester was also prepared as a dioxalate salt using the followingprocedure.

510 mg of the product of Example 1 (0.853 mmol of 96% pure material) wastaken up in 5 ml of H₂O:ACN (1:1) and 1.7 ml of 1M aqueous oxalic acidwas added slowly, while monitoring the pH. The pH was adjusted toapprox. pH 3.0. The clear solution was filtered through a 0.2 micronfilter, frozen and lyophilized to dryness. 150 mg of the lyophilizedmaterial was dissolved in 13.1 ml of 94% IPA/6% H₂O. The mixture wasstirred in a pre-heated 60° C. water bath for 2.5 hours. The heat wasturned off and the mixture was allowed to cool to room temperature. Thevial was refrigerated at 4° C. After 6 days, an oily material wasobserved with what appeared to be a crystal on the side of the vial. Thevial was then allowed to reach room temperature, at which point seeds(synthesis described below) were added and allowed to sit for 16 days.During this time, more crystals were observed to come out of solution.The solid was then filtered and dried using a vacuum pump for 14 hoursto give the dioxalate salt of the title compound (105 mg, 70% yield).

Seed Synthesis

510 mg of the product of Example 1 (0.853 mmol of 96% pure material) wastaken up in 5 ml of H₂O:ACN (1:1) and 1.7 ml of 1M aqueous oxalic acidwas added slowly, while monitoring the pH. The pH was adjusted toapprox. pH 3.0. The clear solution was filtered through a 0.2 micronfilter, frozen and lyophilized to dryness to yield a dioxalate salt.31.5 mg of this dioxalate salt was dissolved in 2.76 ml of 94%IPA/6%H₂0. The mixture was stirred in a pre-heated 60° C. water bath for 2.5hours. After 25 minutes, all of the sample was in solution. The heat wasturned off and the mixture was allowed to cool to room temperature. Thenext day, a small amount of viscous material was present. The vial wasrefrigerated at 4° C. After 4 days, the viscous material was stillpresent. The vial was then placed at room temperature and observed onemonth later. The material appeared to be solid, and was observed to becrystalline under a microscope. The solid was then filtered and driedusing a vacuum pump for 1 hour to give the dioxalate salt (20 mg, 63.5%yield).

Example 1D

Biphenyl-2-ylcarbamic acid1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylester was also prepared as a freebase crystal using the followingprocedure.

230 mg of the product of Example 1 was dissolved in 0.2 ml of H₂O:ACN(1:1), using slight heat. The mixture was then heated in a 70° C. waterbath for 2 hours. The heat was turned off and the mixture was allowed tocool to room temperature, then refrigerated at 4° C. for 1 hour. 50 μlof water was then added (oiled out), followed by the addition of 40 μlof ACN to get the sample back into solution. Seeds (synthesis describedbelow) were added under slow stirring at room temperature. Crystalsstarted to form, and the mixture was allowed to sit overnight, with slowstirring. The next day, a heat cool cycle was applied (30° C. for 10minutes, 40° C. for 10 minutes, then 50° C. for 20 minutes). The heatwas turned off and the mixture allowed to cool overnight, with slowstirring. The next day, a second heat/cool cycle was applied (60° C. for1 hour, with dissolving observed at 70° C.). The heat was turned off andthe mixture allowed to cool overnight, with slow stirring. The next day,crystals were present and a third heat cool cycle was applied (60° C.for 3 hours). The heat was turned off and the mixture allowed to coolovernight, with slow stirring. The next day, a heat cool cycle wasapplied (60° C. for 3 hours, slow cool, then 60° C. for 3 hours). Theheat was turned off and the mixture allowed to cool overnight, with slowstirring. After 3 days, the solid was filtered and placed on a highvacuum line to remove all solvent and give a freebase crystal of thetitle compound.

Seed Synthesis

109 mg of the product of Example 1 was dissolved in 0.56 ml of H₂O:ACN(1:1). The suspension was left in a vial (cap loosely placed on top) toallow for a slower evaporation time. The vial was placed under anitrogen flow environment, although the nitrogen was not used forevaporation, only for the environment. A precipitate was visible within1 day, which was observed to be crystalline under a microscope. Thesolid was then placed on a high vacuum line to remove all solvent togive the freebase crystal. Quantitative recovery, 97.8% pure by HPLC.

Example 1E

Biphenyl-2-ylcarbamic acid1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylester was also prepared as a freebase crystal using the followingalternate procedure.

70 mg of the product of Example 1 was dissolved in 0.1 mL ACN. Afteraddition of 0.3 ml MTBE, the solution appeared cloudy. An additional 50μl of ACN was added to clarify the solution (155 mg/ml ACN:MTBE=1:2).The mixture was left in the vial and capped. A solid appeared by thenext day. The solid was then filtered and placed on a high vacuum lineto remove all solvent and give a freebase crystal of the title compound.

Example 2 Biphenyl-2-ylcarbamic Acid1-(2-{[4-(4-Carbamoylpiperidin-1-ylmethyl)-benzoyl]ethylamino}ethyl)piperidin-4-ylEster

Using the procedure of Example 1, and in Preparation 2 substitutingN-benzyl-N-ethylethanolamine in place of N-benzyl-K-methylethanolamine,the title compound was prepared. MS m/z: [M+H⁺] calcd for C₃₆H₄₅N₅O₄,612.3; found, 612.6.

Preparation 6 Biphenyl-2-ylcarbamic Acid1-(2-{1[4-(4-Methylesterpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylEster

To a three-necked 100 ml flask was added methyl isonipecotate (344 mg,2.4 mmol), acetic acid (136 μl), sodium sulfate (341 mg) and IPA (20ml). The reaction mixture was cooled to 0-10° C. with an ice bath and asolution of the product of Preparation 5 (600 mg, 1.24 mmol) in IPA (10ml) was slowly added. The reaction mixture was stirred at roomtemperature for 1 hour and then cooled to 0-10° C. Sodiumtriacetoxyborohydride (763 mg, 3.6 mmol) was added portion wise. Afterstirring at room temperature for 16 hours, the reaction mixture was thenconcentrated under reduced pressure to a volume of about 5 ml anddiluted with DCM (50 ml). The organic layers were washed with 0.5N HCl(2×30 ml), water (2×30 mL), brine (2×30 ml), dried over sodium sulfate,filtered and concentrated to afford 700 mg of the title compound. (92%yield. MS m/z: [M+H⁺] calcd for C₃₆H₄₄N₄O₅, 612.8; found, 613.5.)

Example 3 Biphenyl-2-ylcarbamic Acid1-(2-{Methyl-[4-(4-methylcarbamoylpiperidin-1-ylmethyl)benzoyl]amino}ethyl)piperidin-4-ylEster

To a 4 ml vial was added the product of Preparation 6 (61.2 mg, 0.1mmol) and methylamine (1 ml, 2M in MeOH). The reaction mixture washeated at 60° C. for 72 hours and was purified by prep HPLC to afford46.9 mg of the title compound. (MS m/z: [M+H⁺] calcd for C₃₆H₄₅N₅O₄,611.8; found, 612.4.)

Example 4 Biphenyl-2-ylcarbamic Acid1-(2-{[4-(4-Ethylcarbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylEster

Using the procedure of Example 3, and substituting ethylamine (1 ml, 2Min EtOH) in place of methylamine (1 ml, 2M in MeOH), 17 mg of the titlecompound was prepared. (MS m/z: [M+H⁺] calcd for C₃₇H₄₇N₅O₄, 625.8;found, 626.4.)

Example 5 Biphenyl-2-ylcarbamic Acid1-(2-{Methyl-[4-(4-propylcarbamoylpiperidin-1-ylmethyl)benzoyl]amino}ethyl)piperidin-4-ylEster

To a 4 ml vial was added the product of Preparation 6 (61.2 mg, 0.1mmol) and propylamine (1 ml). The reaction mixture was heated at 60° C.for 24 hours and was purified by prep HPLC to afford 39.5 mg of thetitle compound. (MS m/z: [M+H⁺] calcd for C₃₈H₄₉N₅O₄, 639.8; found,640.4.)

Example 6 Biphenyl-2-ylcarbamic Acid1-(2-{[4-(4-Isopropylcarbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylEster

Using the procedure of Example 5, and substituting isopropylamine (1 ml)in place of propylamine (1 ml), 27.8 mg of the title compound wasprepared. (MS m/z: [M+H⁺] calcd for C₃₈H₄₉N₅O₄, 639.8; found, 640.4.)

Preparation 7 Biphenyl-2-ylcarbamic Acid1-(2-Boc-aminoethyl)piperidin-4-yl Ester

To a 1-L flask, containing the product of Preparation 2 (25.4 g, 85.6mmol) in DCM (0.43 L) was added DIPEA (29.9 mL, 171.1 mmol) and2-(Boc-Amino) ethyl bromide (21.8 g, 94.4 mmol). The reaction was thenheated to 50° C. overnight (˜18 hours). After overnight, the reactionwas then cooled to 0° C. to induce precipitation of the product. Theprecipitate was filtered and collected to afford the title compound in42% yield (15.8 g). MS m/z: [M+H⁺] calcd for C₂₅H₃₃N₃O₄, 439.3; found,440.4.

Preparation 8 Biphenyl-2-ylcarbamic Acid 1-(2aminoethyl)piperidin-4-ylEster

The product of Preparation 7 (3.5 g, 8.1 mmol) was added to 1:1 DCM:TFA(50 mL) and the reaction was allowed to stir at room temperature for 30minutes. Upon completion, the reaction was diluted with DCM (125 mL) andthe mixture was washed with 1N NaOH (200 mL). The organic layer was thenwashed with water (200 mL), NaCl (sat.) (200 mL), dried over Na₂SO₄ andthen filtered. The solvent was removed under reduced pressure. The crudematerial was sufficiently pure to use without further purification. Thetitle compound was obtained in 94% yield (2.6 g, 7.6 mmol). MS m/z:[M+H⁺] calcd for C₂₀H₂₅N₃O₂, 339.2; found, 339.6.

Preparation 9 2-Fluoro-4-formyl Benzoic Acid

A stirred solution of 4-cyano-2-fluorobenzoic acid (2.5 g, 15.2 mmol) inDCM (100 mL) was cooled to −78° C. and to this was added dropwise DIBAL(30 mL, 45.4 mmol, 25% in toluene), using caution due to H₂ evolution.This was allowed to stir at −78° C. for 4 hours. The reaction wasquenched via addition of MeOH (10 mL), using caution due to H₂evolution. The organic layer was then washed with 1N HCl (100 mL), water(100 mL), NaCl (sat.) (100 mL), dried over MgSO₄ and then filtered. Thesolvent was removed under reduced pressure. The crude material wassufficiently pure to use without further purification. The titlecompound was obtained in 78% yield (2.0 g, 11.9 mmol).

Example 7 Biphenyl-2-ylcarbamic Acid1-{2-[4-(4-Carbamoylpiperidin-1-ylmethyl)-2-fluorobenzoylamino]ethyl}piperidin-4-ylEster

Using the procedure of Example 1 and, in Preparation 5, substituting theproduct of Preparation 8 in place of the product of Preparation 4 andsubstituting the product of Preparation 9 in place of4-carboxybenzaldehyde, the title compound was prepared. MS m/z: [M+H⁺]calcd for C₃₄H₄₀FN₅O₄, 601.7; found, 602.2.

Example 8 Biphenyl-2-ylcarbamic Acid1-(2-{[4-(4-Carbamoylpiperidin-1-ylmethyl)-2-fluorobenzoyl]methylamino}ethyl)piperidin-4-ylEster

Using the procedure of Example 1 and, in Preparation 5, substituting theproduct of Preparation 9 in place of 4-carboxybenzaldehyde, the titlecompound was prepared. MS m/z: [M+H⁺] calcd for C₃₅H₄₂FN₅O₄, 615.8;found, 616.2

Preparation 10 Piperidine-4-carboxylic Acid Diethylamide

To a stirred solution of 1-tert-butoxycarbonylpiperidine-4-carboxylicacid (5 g, 22.0 mmol) in DMF (100 mL) was added diethyl amine (4.6 mL,44 mmol), triethylamine (9.1 mL, 66.0 mmol), HOAt (22.0 mL, 0.5 M inDMF, 22.0 mmol) and finally EDCI (8.4 g, 44 mmol). This was allowed tostir for 14 hours at room temperature. The solvent was then removedunder reduced pressure. The mixture was taken up in DCM (100 mL). Theorganic layer was then washed with water (100 mL), 1N HCl (100 mL), NaCl(sat.) (100 mL), dried over MgSO₄ and then filtered. To the organiclayer was added TFA (33 mL). The reaction was allowed to stir at roomtemperature for 2 hours. The solvent was then removed under reducedpressure. The mixture was taken up in DCM (100 mL). The organic layerwas then washed with 1N NaOH (100 mL), water (100 mL), NaCl (sat.) (100mL), dried over MgSO₄ and then filtered. The solvent was removed underreduced pressure. The crude material was sufficiently pure to usewithout further purification. The title compound was obtained in 86%yield (3.5 g, 19.0 mmol).

Example 9 Biphenyl-2-ylcarbamic Acid1-{2-[4-(4-Diethylcarbamoylpiperidin-1-ylmethyl)-2-fluorobenzoylamino]ethyl}piperidin-4-ylEster

Using the procedure of Example 1 and, in Preparation 5, substituting theproduct of Preparation 9 in place of 4-carboxybenzaldehyde andsubstituting the product of Preparation 8 in place of the product ofPreparation 4 and, in Example 1, substituting the product of Preparationto in place of isonipecotamide, the title compound was prepared. MS m/z:[M+H⁺] calcd for C₃₈H₄₈FN₅O₄, 657.8; found, 658.4.

Example 10 Biphenyl-2-ylcarbamic Acid1-(2-{[4-(4-Diethylcarbamoylpiperidin-1-ylmethyl)-2-fluorobenzoyl]methylamino}ethyl)piperidin-4-ylEster

Using the procedure of Example 1 and, in Preparation 5, substituting theproduct of Preparation 9 in place of 4-carboxybenzaldehyde and, inExample 1, substituting the product of Preparation 10 in place ofisonipecotamide the title compound was prepared. MS m/z: [M+H⁺] calcdfor C₃₉H₅₀FN₅O₄, 671.9; found, 672.4.

Example 11 Biphenyl-2-ylcarbamic Acid1-(2-{[4-(4-Diethylcarbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylEster

Using the procedure of Example 1 but substituting the product ofPreparation 10 in place of isonipecotamide, the title compound wasprepared. MS m/z: [M+H⁺] calcd for C₃₉H₅₁N₅O₄, 653.9; found, 654.4.

Example 12 Biphenyl-2-ylcarbamic Acid1-(2-{[4-(3-(S)-Diethylcarbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylEster

Using the procedure of Example 1 but substitutingpiperidine-3-(S)-carboxylic acid diethylamide in place ofisonipecotamide, the title compound was prepared. MS m/z: [M+H⁺] calcdfor C₃₉H₅₁N₅O₄, 653.9; found, 654.4.

The preparation of piperidine-3-(S)-carboxylic acid diethylamide wasdone according to Chirality 7(2): 90-95 (1995).

Preparation 11N-{2-[4-(Biphenyl-2-ylcarbamoyloxy)piperidin-1-yl]ethyl}-2,5-dibromo-N-methylterephthalamicAcid

To a 100 mL flask containing the product of Preparation 2 (2.5 g, 7.1mmol) in DMF (20 mL) was added 2,5-dibromoterephthalic acid (6.88 g,21.2 mmol) followed by DIPEA (1.6 mL, 9.2 mmol) and HATU (3.23 g, 8.5mol). The yellow slurry was stirred at room temperature for 3 hours (allmaterial in solution following completion of reaction). The reactionmixture was diluted with DCM (200 mL). To the solution was added 1N NaOH(150 mL) and MeOH (minimal amount added in order to dissolve the finewhite precipitate that was observed following the addition of base). Thesolution was transferred to a separatory funnel and the aqueous layerdiscarded. The organic layer was washed with 1N HCl (1×150 mL), driedover sodium sulfate, filtered and the solvent removed under reducedpressure to provide 7 g of the title compound (>100% yield due to thepresence of residual DMF). This material was used without furtherpurification. MS m/z: [M+H⁺] calcd f for C₂₉H₂₉Br₂N₃O₅, 659.4; found,660.3. R_(f)=3.39 min(2-90 ACN: H₂O, reverse phase HPLC).

Preparation 12N-{2-[4-(Biphenyl-2-ylcarbamoyloxy)piperidin-1-yl]ethyl}-2,5-dibromo-N-methylterephthalamicAcid Methyl Ester

To a 100 mL flask containing the product of Preparation 11 (7.0 g, 10.6mmol) was added a solution of toluene/MeOH (9:1, 70 mL). All of thesolid material did not dissolve, so an additional 3 mL of MeOH wasadded. The solution was cooled to 0° C. over an ice bath andtrimethylsilyldiazomethane (2.0M solution in hexanes, 6.3 mL, 12.7 mmol)was added via syringe. The reaction mixture was allowed to warm to roomtemperature. After 2 hours stirring, HPLC and MS analysis indicated thatthe reaction was not complete. Additional trimethylsilyldiazomethane(10.0 mL) was added and the reaction was stirred at room temperature for70 hours. Although HPLC analysis had indicated that the reaction was notcomplete, acetic acid (15 mL) was added to the reaction mixture and theresulting solution was concentrated under reduced pressure. The crudeproduct was purified by silica gel chromatography using a gradient of 2%to 5% MeOH in DCM as the eluent to provide 2.32 g of the title compound(49% yield). MS m/z: [M+H⁺] calcd f for C₃₀H₃₁Br₂N₃O₅, 673.4; found,674.3. R_(f)=4.26 min (2-90 ACN: H₂O, reverse phase HPLC).

Preparation 13 Biphenyl-2-yl-carbamic Acid1-{2-[(2,5-Dibromo-4-hydroxymethylbenzoyl)methylamino]ethyl}piperidin-4-ylEster

To a 100 mL flask containing the product of Preparation 12 (2.2 g, 3.3mmol) was added THF (35 mL). Using an ice bath, the mixture was cooledto about 0° C. and lithium aluminum hydride (11.0M solution in THF, 6.6mL, 6.6 mmol) was added via syringe. The resulting slurry was stirred atroom temperature for 4 h. The reaction was quenched by adding 1N NaOH(100 mL). The aqueous layer was separated and the organic layer waswashed with brine (50 mL), dried over sodium sulfate, filtered andconcentrated under reduced pressure (80.2% pure by HPLC). A portion ofthe crude product was purified by preparatory HPLC (20-40 ACN: H₂O,reverse phase HPLC) to afford 317 mg of the TFA salt. The TFA salt ofthe desired product was partitioned between EtOAc (10 mL) and saturatedsodium bicarbonate solution (10 mL). The organic layer was washed withbrine (5 mL), dried over sodium sulfate, filtered and concentrated toprovide 223.6 mg of the title compound. MS m/z: [M+H⁺] calcd forC₂₉H₃₁Br₂N₃O₄, 645.4; found, 646.3. R_(f)=3.56 min (10-70 ACN: H₂O,reverse phase HPLC).

Preparation 14 Methanesulfonic Acid4-({2-[4-(Biphenyl-2-ylcarbamoyloxy)piperidin-1-yl]ethyl}methylcarbamoyl)-2,5-dibromobenzylEster

To a 25 mL flask containing the product of Preparation 13 (223.6 mg,0.346 mmol) was added DCM (10 mL) followed by DIPEA (135.5 uL, 0.778mmol) and methanesulfonyl chloride (41 uL, 0.528 mmol). The reaction wasstirred for 30 minutes at room temperature. To the reaction mixture wasthen added saturated sodium bicarbonate solution (10 mL). The aqueouslayer was discarded and the organic layer was washed with brine (5 mL),dried over sodium sulfate, filtered and concentrated under reducedpressure to afford 229 mg of the title compound (91% yield). MS m/z:[M+H⁺] calcd for C₃₀H₃₃Br₂N₃O₆S, 723.5; found, 724.3. R_(f)=3.77 min(10-70 ACN: H₂O, reverse phase HPLC).

Example 13 Biphenyl-2-ylcarbamic Acid1-(2-{[2,5-dibromo4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylEster

To a 25 mL flask containing the product of Preparation 14 (229 mg, 0.316mmol) was added isonipecotamide (48.7 mg, 0.380 mmol), DIPEA (110.2 uL,0.633 mmol), and ACN (4 mL). The reaction mixture was stirred at roomtemperature for 63 hours under a nitrogen atmosphere. The reactionmixture was then diluted with DCM (15 mL) and washed with saturatedaqueous sodium bicarbonate solution. The product was extracted into theaqueous layer using 1.0 N HCl (2×10 mL). The aqueous layer was washedwith DCM (2×15 mL) and the pH was adjusted to 10-11 using 1.0 N NaOH.This mixture was then extracted with DCM (3×20 mL) and the combinedorganic layers were washed with brine (10 mL), dried over sodiumsulfate, filtered and concentrated to provide the title compound. MSm/z: [M+H⁺] calcd for C₃₅H₄₁Br₂N₅O₄, 756.5; found, 756.3. R_(f)=2.65 min(10-70 ACN: H₂O, reverse phase HPLC).

Example 14 Biphenyl-2-yl-carbamic Acid1-(2-{[14-(2-Carbamoyl-piperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylEster

The title compound was prepared using the procedures described inExample 1, and substituting the appropriate starting materials. MS m/z:[M+H⁺] calcd for C₃₅H₄₃N₅O₄, 598.3; found, 597.8.

Example 15 Biphenyl-2-yl-carbamic Acid1-(2-{[4-(4-Carbamoyl-piperidin-1-ylmethyl)-2-methoxybenzoyl]methylamino}ethyl)piperidin-4-ylEster

To a stirred solution of 4-bromo-3-methoxy-benzoic acid (15.0 g, 58mmol) in DMSO (150 mL) was added NaHCO₃ (20.0 g, 230 mmol). This washeated to 80° C. for 18 hours. The reaction was then cooled to roomtemperature and the solvent removed under reduced pressure. The crudereaction mixture was then dissolved in DCM (200 mL) and washed with 1NHCl (100 mL), water (100 mL), NaCl (sat.) (100 mL), dried over MgSO₄ andthen filtered. The solvent was removed under reduced pressure. The crudematerial was sufficiently pure to use without further purification. Theproduct, 4-formyl-3-methoxybenzoic acid methyl ester, was obtained in79% yield (8.9 g, 45.8 mmol).

To a stirred solution of 4-formyl-3-methoxy-benzoic acid methyl ester(5.0 g, 26 mmol) in tert-butyl alcohol (200 mL) was added NaH₂PO₄-2H₂O(3.6 g, 26 mmol), water (50 mL), 2-methyl-2-butene (11 mL, 104 mmol),and finally NaClO₂ (7.02 g, 78 mmol). The reaction was allowed to stirat room temperature for 4 hours. The solvent was then removed underreduced pressure. The crude reaction mixture was then dissolved in DCM(200 mL) and the product was extracted with 1N NaOH (200 mL). Theaqueous layer was washed with DCM (200 mL) and then neutralized with 6NHCl (˜40 mL) and the product extracted with DCM (200 mL). The organiclayer was then washed with water (100 mL), NaCl (sat.) (100 mL), driedover MgSO₄ and then filtered. The solvent was removed under reducedpressure. The crude material was sufficiently pure to use withoutfurther purification. The product, 2-methoxyterephthalic acid 4-methylester, was obtained in 47% yield (2.4 g, 12.3 mmol).

To a stirred solution of 2-methoxyterephthalic acid 4-methyl ester (450mg, 2.1 mmol) in DMF (10 mL) was added EDC (630 mg, 3.3 mmol), HOAt (2.4mL, 1.18 mmol, 0.5M in DMF) and DIPEA (1.3 mL, 7.05 mmol). When themixture was homogenous, a solution of the product of Preparation 4 (830mg, 2.4 mmol) was added slowly. The reaction mixture was stirred at roomtemperature for 16 hours and then washed with water (100 mL), 1N HCl(100 mL), 1N NaOH (100 mL), brine (100 mL), dried over MgSO₄, filteredand concentrated to afford an ester product in 89% yield (1.04 g, 1.9mmol). MS m/z: [M+H⁺] calcd for C₃₁H₃₅N₃O₆, 545.6; found, 546.6.

To a stirred solution of this ester product (1.0 g, 1.8 mmol) in THF(100 mL) at 0° C., was added methanol (57 μL, 1.8 mmol), followed byLiAlH₄ (1.8 mL, 1.8 mmol, 1.0M in THF) was added. The ice bath wasremoved, and the reaction mixture was stirred at room temperature for 1hour. The reaction was quenched with 1N HCl (aq) at 0° C. until no morebubbling, stirring was continued for 10 minutes. The solvent was removedunder reduced pressure. The crude reaction mixture was taken up in DCM(100 mL) and washed with water (100 mL), NaCl (sat.) (100 mL), driedover MgSO₄ and then filtered. The solvent was removed under reducedpressure. The crude material was sufficiently pure to use withoutfurther purification. The alcohol product was obtained in 89% yield (831mg, 1.6 mmol). MS m/z: [M+H⁺] calcd for C₃₀H₃₅N₃O₅, 517.6; found, 518.6.

To a stirred solution of this alcohol product (78 mg, 1.5 mmol) in DCM(2.5 mL) at −15° C. was added DMSO (130 μL, 22.5 mmol), DIPEA (130 μL,7.5 mmol). To the solution was added sulfur trioxide-pyridine complex(240 mg, 15 mmol). After 30 minutes, the reaction mixture was quenchedwith H₂O (˜3 mL). Two layers were separated, the organic layer was driedover MgSO₄, filtered and the aldehyde product was used directly in thenext reaction.

Using the procedure of Example 1 but substituting this aldehyde productin place of the product of Preparation 5, the title compound wasprepared. MS m/z: [M+H⁺] calcd for C₃₆H₄₅N₅O₅, 627.3; found, 628.2.

Using the procedures described herein and substituting the appropriatestarting materials, the following compounds were prepared:

Example 16

Biphenyl-2-yl-carbamic acid1-(2-{[5-(4-carbamoylpiperidin-1-ylmethyl)thiophene-2-carbonyl]methylamino}ethyl)piperidin-4-ylester. MS m/z: [M+H⁺] calcd for C₃₃H₄₁N₅O₄S, 604.3; found 604.2.

Example 17

Biphenyl-2-yl-carbamic acid1-(2-{[5-((R)-3-diethylcarbamoylpiperidin-1-ylmethyl)thiophene-2-carbonyl]methylamino}ethyl)piperidin-4-ylester. MS m/z: [M+H⁺] calcd for C₃₇H₄₉N₅O₄S, 660.4; found 660.4.

Example 18

Biphenyl-2-yl-carbamic acid1-(2-{[5-((R)-3-diethylcarbamoylpiperidin-1-ylmethyl)thiophene-2-carbonyl]amino}ethyl)piperidin-4-ylester. MS m/z: [M+H⁺] calcd for C₃₆H₇N₅O₄S, 646.3; found 646.4.

Example 19

Biphenyl-2-yl-carbamic acid1-(2-{[5-(4-carbamoylpiperidin-1-ylmethyl)thiophene-2-carbonyl]amino}ethyl)piperidin-4-ylester. MS m/z: [M+H⁺] calcd for C₃₂H₃₉N₅O₄S, 590.3; found 590.2.

Example 20

biphenyl-2-yl-carbamic acid 1-(2-{[5-((R)-3-diethylcarbamoylpiperidin-1-ylmethyl)-1H-pyrrole-2-carbonyl]methylamino}ethyl)piperidinylester. MS m/z: [M+H⁺] calcd for C₃₇H₅₀N₆O₄, 643.4; found 643.2.

Example 21

Biphenyl-2-yl-carbamic acid1-(2-{[5-(4-carbamoylpiperidin-1-ylmethyl)-1H-pyrrole-2-carbonyl]methylamino}ethyl)piperidin-4-ylester. MS m/z: [M+H⁺] calcd for C₃₃H₄₂N₆O₄, 587.3; found 587.2.

Example 22

Biphenyl-2-yl-carbamic acid 1-(2-{[5-((R)-3-diethylcarbamoylpiperidin-1-ylmethyl)furan-2-carbonyl]methylamino}ethyl)piperidin-4-ylester. MS m/z: [M+H⁺] calcd for C₃₇H₄₉N₅O₅, 644.4; found 644.4.

Example 23

Biphenyl-2-yl-carbamic acid1-(2-{[5-(4-diethylcarbamoyl-piperidin-1-ylmethyl)furan-2-carbonyl]methylamino}ethyl)piperidin-4-ylester. MS m/z: [M+H⁺] calcd for C₃₇H₄₉N₅O₅, 644.4; found 644.4.

Example 24

Biphenyl-2-yl-carbamic acid1-(2-{[5-(4-carbamoylpiperidin-1-ylmethyl)furan-2-carbonyl]-amino}ethyl)piperidin-4-ylester. MS m/z: [M+H⁺] calcd for C₃₂H₃₉N₅O₅, 574.3; found 574.2.

Example 25

Biphenyl-2-yl-carbamic acid 1-(2-{[5-((R)-3-diethylcarbamoylpiperidin-1-ylmethyl)furan-2-carbonyl]amino}ethyl)piperidin-4-yl ester.MS m/z: [M+H⁺] calcd for C₃₆H₄₇N₅O₅, 630.4.

Example 26

Biphenyl-2-yl-carbamic acid1-[2-({3-[4-(3-carbamoylpiperidin-1-ylmethyl)phenyl]propionyl}methylamino)ethyl]piperidin-4-ylester. MS m/z: [M+H⁺] calcd for C₃₇H₄₇N₅O₄, 626.4; found 625.8.

Example 27

Biphenyl-2-yl-carbamic acid1-[2-({3-[4-(4-carbamoylpiperidin-1-ylmethyl)phenyl]propionyl}methylamino)ethyl]piperidin-4-ylester. MS m/z: [M+H⁺] calcd for C₃₇H₇N₅O₄, 626.4; found 625.8.

Example 28

Biphenyl-2-yl-carbamic acid1-(2-{3-[4-(4-carbamoylpiperidin-1-ylmethyl)phenyl]propionylamino}ethyl)piperidin-4-ylester. MS m/z: [M+H⁺] calcd for C₃₆H₄₅N₅O₄, 612.4; found 611.8.

Example 29

Biphenyl-2-yl-carbamic acid 1-(2-{3-[4-(4-diethylcarbamoylpiperidin-1-ylmethyl)phenyl]propionylamino}ethyl)piperidin-4-yl ester.MS m/z: [M+H⁺] calcd for C₄₀H₅₃N₅O₄, 668.4; found 667.9.

Example 30

Biphenyl-2-yl-carbamic acid 1-(2-{3-[4-(3-diethylcarbamoylpiperidin-1-ylmethyl)phenyl]propionylamino}ethyl)piperidin-4-yl ester.MS m/z: [M+H⁺] calcd for C₄₀H₅₃N₅O₄, 668.4; found 667.9.

Using the procedures described herein and substituting the appropriatestarting materials, the following compounds can be prepared:

Example 31

Biphenyl-2-ylcarbamic acid1-{2-[4-(4-carbamoyl-piperidin-1-ylmethyl)benzoylamino]ethyl}piperidin-4-ylester;

Example 32

Biphenyl-2-ylcarbamic acid1-{2-([4-(4-carbamoylpiperidin-1-ylmethyl)-2-chloro-benzoyl]methylamino}ethyl)piperidin-4-ylester;

Example 33

Biphenyl-2-ylcarbamic acid1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)-2-chloro-5-methoxybenzoyl]methylamino}ethyl)piperidin-4-ylester; and

Example 34

Biphenyl-2-ylcarbamic acid1-[2-({2-[4-(4-carbamoylpiperidin-1-ylmethyl)phenyl]acetyl}methylamino)ethyl]piperidin-4-ylester

Assay 1 Radioligand Binding Assay

A. Membrane Preparation from Cells Expressing hM₁, hM₂, hM₃, and hM₄Muscarinic Receptor Subtypes

CHO cell lines stably expressing cloned human hM₁, hM₂, hM₃ and hM₄muscarinic receptor subtypes, respectively, were grown to nearconfluency in medium consisting of HAM's F-12 supplemented with 10% FBSand 250 μg/mL Geneticin. The cells were grown in a 5% CO₂, 37° C.incubator and lifted with 2 mM EDTA in dPBS. Cells were collected by 5minute centrifugation at 650×g, and cell pellets were either storedfrozen at −80° C. or membranes were prepared immediately. For membranepreparation, cell pellets were resuspended in lysis buffer andhomogenized with a Polytron PT-2100 tissue disrupter (Kinematica AG; 20seconds×2 bursts). Crude membranes were centrifuged at 40,000×g for 15minutes at 4° C. The membrane pellet was then resuspended withresuspension buffer and homogenized again with the Polytron tissuedisrupter. The protein concentration of the membrane suspension wasdetermined by the method described in Lowry, O. et al., Journal ofBiochemistry 193:265 (1951). All membranes were stored frozen inaliquots at −80° C. or used immediately. Aliquots of prepared hM₅receptor membranes were purchased directly from Perkin Elmer and storedat −80° C. until use.

B. Radioligand Binding Assay on Muscarinic Receptor Subtypes hM₁, hM₂,hM₃, hM₄ and hM₅

Radioligand binding assays were performed in 96-well microtiter platesin a total assay volume of 100 μL. CHO cell membranes stably expressingeither the hM₁, hM₂, hM₃, hM4 or hM₅ muscarinic subtype were diluted inassay buffer to the following specific target protein concentrations(μg/well): 10 μg for hM₁, 10-15 μg for hM₂, 10-20 μg for hM₃, 10-20 μgfor hM₄, and 10-12 μg for hM₅. The membranes were briefly homogenizedusing a Polytron tissue disruptor (10 seconds) prior to assay plateaddition. Saturation binding studies for determining K_(D) values of theradioligand were performed using L-[N-methyl-³H]scopolamine methylchloride ([³H]-NMS) (TRK666, 84.0 Ci/mmol, Amersham Pharmacia Biotech,Buckinghamshire, England) at concentrations ranging from 0.001 nM to 20nM. Displacement assays for determination of K_(i) values of testcompounds were performed with [3H]-NMS at 1 nM and eleven different testcompound concentrations. The test compounds were initially dissolved toa concentration of 400 μM in dilution buffer and then serially diluted5× with dilution buffer to final concentrations ranging from 10 pM to100 μM. The addition order and volumes to the assay plates were asfollows: 25 μL radioligand, 25 μL diluted test compound, and 50 μLmembranes. Assay plates were incubated for 60 minutes at 37° C. Bindingreactions were terminated by rapid filtration over GF/B glass fiberfilter plates (PerkinElmer Inc., Wellesley, Mass.) pre-treated in 1%BSA. Filter plates were rinsed three times with wash buffer (10 mMHEPES) to remove unbound radioactivity. Plates were then air dried, and50 μL Microscint-20 liquid scintillation fluid (PerkinElmer Inc.,Wellesley, Mass.) was added to each well. The plates were then countedin a PerkinElmer Topcount liquid scintillation counter (PerkinElmerInc., Wellesley, Mass.). Binding data were analyzed by nonlinearregression analysis with the GraphPad Prism Software package (GraphPadSoftware, Inc., San Diego, Calif.) using the one-site competition model.K_(i) values for test compounds were calculated from observed IC₅₀values and the K_(D) value of the radioligand using the Cheng-Prusoffequation (Cheng Y; Prusoff W. H. Biochemical Pharmacology22(23):3099-108 (1973)). K_(i) values were converted to a K_(i) valuesto determine the geometric mean and 95% confidence intervals. Thesesummary statistics were then converted back to K_(i) values for datareporting.

In this assay, a lower K_(i) value indicates that the test compound hasa higher binding affinity for the receptor tested. For example, thecompounds of Examples 1 and 2 were found to have a K_(i) value of lessthan about 5 nM for the M₃ muscarinic receptor subtype in this assay.

Assay 2 Muscarinic Receptor Functional Potency Assays

A. Blockade of Agonist-Mediated Inhibition of cAMP Accumulation

In this assay, the functional potency of a test compound was determinedby measuring the ability of the test compound to blockoxotremorine-inhibition of forskolin-mediated cAMP accumulation inCHO-K1 cells expressing the hM₂ receptor.

cAMP assays were performed in a radioimmunoassay format using theFlashplate Adenylyl Cyclase Activation Assay System with ¹²⁵I-cAMP (NENSMP004B, PerkinElmer Life Sciences Inc., Boston, Mass.), according tothe manufacturer's instructions.

Cells were rinsed once with dPBS and lifted with Trypsin-EDTA solution(0.05% trypsin/0.53 mM EDTA) as described in the Cell Culture andMembrane Preparation section above. The detached cells were washed twiceby centrifugation at 650×g for five minutes in 50 mLs dPBS. The cellpellet was then re-suspended in 10 mL dPBS, and the cells were countedwith a Coulter Z1 Dual Particle Counter (Beckman Coulter, Fullerton,Calif.). The cells were centrifuged again at 650×g for five minutes andre-suspended in stimulation buffer to an assay concentration of1.6×10⁶−2.8×10⁶ cells/mL.

The test compound was initially dissolved to a concentration of 400 μMin dilution buffer (dPBS supplemented with 1 mg/mL BSA (0.1%)), and thenserially diluted with dilution buffer to final molar concentrationsranging from 100 μM to 0.1 nM. Oxotremorine was diluted in a similarmanner.

To measure oxotremorine inhibition of AC activity, 25 μL forskolin (25μM final concentration diluted in dPBS), 25 μL diluted oxotremorine, and50 μL cells were added to agonist assay wells. To measure the ability ofa test compound to block oxotremorine-inhibited AC activity, 25 μLforskolin and oxotremorine (25 μM and 5 μM final concentrations,respectively, diluted in dPBS), 25 μL diluted test compound, and 50 μLcells were added to remaining assay wells.

Reactions were incubated for 10 minutes at 37° C. and stopped byaddition of 100 μL ice-cold detection buffer. Plates were sealed,incubated overnight at room temperature and counted the next morning ona PerkinElmer TopCount liquid scintillation counter (PerkinElmer Inc.,Wellesley, Mass.). The amount of cAMP produced (pmol/well) wascalculated based on the counts observed for the samples and cAMPstandards, as described in the manufacturer's user manual. Data wereanalyzed by nonlinear regression analysis with the GraphPad PrismSoftware package (GraphPad Software, Inc., San Diego, Calif.) using thenon-linear regression, one-site competition equation. The Cheng-Prusoffequation was used to calculate the K_(i), using the EC₅₀ of theoxotremorine concentration-response curve and the oxotremorine assayconcentration as the K_(D) and [L], respectively. The K_(i) values wereconverted to pK_(i) values to determine the geometric mean and 95%confidence intervals. These summary statistics were then converted backto K_(i) values for data reporting.

In this assay, a lower K_(i) value indicates that the test compound hasa higher functional activity at the receptor tested. Exemplary compoundsof the invention that were tested in this assay, typically were found tohave a K_(i) value of less than about 10 nM for blockade ofoxotremorine-inhibition of forskolin-mediated cAMP accumulation inCHO-K1 cells expressing the hM₂ receptor. For example, the compound ofExample 1 was found to have a K_(i) value of less than about 5 nM.

B. Blockade of Agonist-Mediated [³⁵S]GTPγS-Binding

In a second functional assay, the functional potency of test compoundscan be determined by measuring the ability of the compounds to blockoxotremorine-stimulated [³⁵S]GTPγS-binding in CHO-K1 cells expressingthe hM₂ receptor.

At the time of use, frozen membranes were thawed and then diluted inassay buffer with a final target tissue concentration of 5-10 μg proteinper well. The membranes were briefly homogenized using a PolytronPT-2100 tissue disrupter and then added to the assay plates.

The EC₉₀ value (effective concentration for 90% maximal response) forstimulation of [³⁵S]GTPγS binding by the agonist oxotremorine wasdetermined in each experiment.

To determine the ability of a test compound to inhibitoxotremorine-stimulated [³⁵S]GTPγS binding, the following was added toeach well of 96 well plates: 25 μL of assay buffer with [³⁵S]GTPγS (0.4nM), 25 μL of oxotremorine (EC₉₀) and GDP (3 μM), 25 μL of diluted testcompound and 25 μL CHO cell membranes expressing the hM₂ receptor. Theassay plates were then incubated at 37° C. for 60 minutes. The assayplates were filtered over 1% BSA-pretreated GF/B filters using aPerkinElmer 96-well harvester. The plates were rinsed with ice-cold washbuffer for 3×3 seconds and then air or vacuum dried. Microscint-20scintillation liquid (50 μL) was added to each well, and each plate wassealed and radioactivity counted on a topcounter (PerkinElmer). Datawere analyzed by nonlinear regression analysis with the GraphPad PrismSoftware package (GraphPad Software, Inc., San Diego, Calif.) using thenon-linear regression, one-site competition equation. The Cheng-Prusoffequation was used to calculate the K_(i), using the IC. % values of theconcentration-response curve for the test compound and the oxotremorineconcentration in the assay as the K_(D) and [L], ligand concentration,respectively.

In this assay, a lower K_(i) value indicates that the test compound hasa higher functional activity at the receptor tested. Exemplary compoundsof the invention that were tested in this assay, typically were found tohave a K_(i) value of less than about 10 nM for blockade ofoxotremorine-stimulated [³⁵S]GTPγS-binding in CHO-K1 cells expressingthe hM₂ receptor. For example, the compound of Example 1 was found tohave a K_(i) value of less than about 5 nM.

C. Blockade of Agonist-Mediated Calcium Release Via FLIPR Assays

Muscarinic receptor subtypes (M₁, M₃ and M₅ receptors), which couple toG_(q) proteins, activate the phospholipase C (PLC) pathway upon agonistbinding to the receptor. As a result, activated PLC hydrolyzesphosphatyl inositol diphosphate (PIP₂) to diacylglycerol (DAG) andphosphatidyl-1,4,5-triphosphate (IP₃), which in turn generates calciumrelease from intracellular stores, i.e., endoplasmic and sarcoplasmicreticulum. The FLIPR (Molecular Devices, Sunnyvale, Calif.) assaycapitalizes on this increase in intracellular calcium by using a calciumsensitive dye (Fluo-4AM, Molecular Probes, Eugene, Oreg.) thatfluoresces when free calcium binds. This fluorescence event is measuredin real time by the FLIPR, which detects the change in fluorescence froma monolayer of cells cloned with human M₁ and M₃, and chimpanzee M₅receptors. Antagonist potency can be determined by the ability ofantagonists to inhibit agonist-mediated increases in intracellularcalcium.

For FLIPR calcium stimulation assays, CHO cells stably expressing thehM₁, hM₃ and cM₅ receptors are seeded into 96-well FLIPR plates thenight before the assay is done. Seeded cells are washed twice byCellwash (MTX Labsystems, Inc.) with FLIPR buffer (10 mM HEPES, pH 7.4,2 mM calcium chloride, 2.5 mM probenecid in HBSS without calcium andmagnesium) to remove growth media and leaving 50 μL/well of FLIPRbuffer. The cells are then incubated with 50 μL/well of 4 μM FLUO-4AM (a2× solution was made) for 40 minutes at 37° C., 5% carbon dioxide.Following the dye incubation period, cells are washed two times withFLIPR buffer, leaving a final volume of 50 μL/well.

To determine antagonist potency, the dose-dependent stimulation ofintracellular Ca²⁺ release for oxotremorine is first determined so thatantagonist potency can later be measured against oxotremorinestimulation at an EC₉₀ concentration. Cells are first incubated withcompound dilution buffer for 20 minutes, followed by agonist addition,which is performed by the FLIPR. An EC₉₀ value for oxotremorine isgenerated according to the method detailed in the FLIPR measurement anddata reduction section below, in conjunction with the formulaEC_(F)=((F/100-F)ˆ1/H)*EC₅₀. An oxotremorine concentration of 3×EC_(F)is prepared in stimulation plates such that an EC₉₀ concentration ofoxotremorine is added to each well in the antagonist inhibition assayplates.

The parameters used for the FLIPR are: exposure length of 0.4 seconds,laser strength of 0.5 watts, excitation wavelength of 488 nm, andemission wavelength of 550 nm. Baseline is determined by measuring thechange in fluorescence for 10 seconds prior to addition of agonist.Following agonist stimulation, the FLIPR continuously measured thechange of fluorescence every 0.5 to 1 second for 1.5 minutes to capturethe maximum fluorescence change.

The change of fluorescence is expressed as maximum fluorescence minusbaseline fluorescence for each well. The raw data is analyzed againstthe logarithm of drug concentration by nonlinear regression withGraphPad Prism (GraphPad Software, Inc., San Diego, Calif.) using thebuilt-in model for sigmoidal dose-response. Antagonist K_(i) values aredetermined by Prism using the oxotremorine EC₅₀ value as the K_(D) andthe oxotremorine EC₉₀ for the ligand concentration according to theCheng-Prusoff equation (Cheng & Prusoff, 1973).

In this assay, a lower K_(i) value indicates that the test compound hasa higher functional activity at the receptor tested. Exemplary compoundsof the invention that were tested in this assay, typically were found tohave a K value of less than about 10 nM for blockade of agonist-mediatedcalcium release in CHO cells stably expressing the hM₃ receptor. Forexample, the compound of Example 1 was found to have a K_(i) value ofless than about 5 nM for the hM₃ receptor.

Assay 3 Determination of Duration of Bronchoprotection in Guinea PigModel of Acetylcholine-Induced Bronchoconstriction

This in vivo assay was used to assess the bronchoprotective effects oftest compounds exhibiting muscarinic receptor antagonist activity.

Groups of six male guinea pigs (Duncan-Hartley (HsdPoc:DH) Harlan,Madison, Wis.) weighing between 250 and 350 g were individuallyidentified by cage cards. Throughout the study animals were allowedaccess to food and water ad libitum.

Test compounds were administered via inhalation over 10 minutes in awhole-body exposure dosing chamber (R&S Molds, San Carlos, Calif.). Thedosing chambers were arranged so that an aerosol was simultaneouslydelivered to 6 individual chambers from a central manifold. Guinea pigswere exposed to an aerosol of a test compound or vehicle (WFI). Theseaerosols were generated from aqueous solutions using an LC StarNebulizer Set (Model 22F51, PARI Respiratory Equipment, Inc. Midlothian,Va.) driven by a mixture of gases (CO₂=5%, O₂=21% and N₂=74%) at apressure of 22 psi. The gas flow through the nebulizer at this operatingpressure was approximately 3 L/minute. The generated aerosols weredriven into the chambers by positive pressure. No dilution air was usedduring the delivery of aerosolized solutions. During the 10 minutenebulization, approximately 1.8 mL of solution was nebulized. This wasmeasured gravimetrically by comparing pre- and post-nebulization weightsof the filled nebulizer.

The bronchoprotective effects of test compounds administered viainhalation were evaluated using whole body plethysmography at 1.5, 24,48 and 72 hours post-dose.

Forty-five minutes prior to the start of the pulmonary evaluation, eachguinea pig was anesthetized with an intramuscular injection of ketamine(43.75 mg/kg), xylazine (3.50 mg/kg) and acepromazine (1.05 mg/kg).After the surgical site was shaved and cleaned with 70% alcohol, a 2-3cm midline incision of the ventral aspect of the neck was made. Then,the jugular vein was isolated and cannulated with a saline-filledpolyethylene catheter (PE-50, Becton Dickinson, Sparks, Md.) to allowfor intravenous infusions of ACh (Sigma-Aldrich, St. Louis, Mo.) insaline. The trachea was then dissected free and cannulated with a 14Gteflon tube (#NE-014, Small Parts, Miami Lakes, Fla.). If required,anesthesia was maintained by additional intramuscular injections of theaforementioned anesthetic mixture. The depth of anesthesia was monitoredand adjusted if the animal responded to pinching of its paw or if therespiration rate was greater than 100 breaths/minute.

Once the cannulations were complete, the animal was placed into aplethysmograph (#PLY3114, Buxco Electronics, Inc., Sharon, Conn.) and anesophageal pressure cannula (PE-160, Becton Dickinson, Sparks, Md.) wasinserted to measure pulmonary driving pressure (pressure). The teflontracheal tube was attached to the opening of the plethysmograph to allowthe guinea pig to breathe room air from outside the chamber. The chamberwas then sealed. A heating lamp was used to maintain body temperatureand the guinea pig's lungs were inflated 3 times with 4 mL of air usinga 10 mL calibration syringe (#5520 Series, Hans Rudolph, Kansas City,Mo.) to ensure that the lower airways did not collapse and that theanimal did not suffer from hyperventilation.

Once it was determined that baseline values were within the range0.3-0.9 mL/cm H₂O for compliance and within the range 0.1-0.199 cmH₂O/mL per second for resistance, the pulmonary evaluation wasinitiated. A Buxco pulmonary measurement computer program enabled thecollection and derivation of pulmonary values.

Starting this program initiated the experimental protocol and datacollection. The changes in volume over time that occur within theplethysmograph with each breath were measured via a Buxco pressuretransducer. By integrating this signal over time, a measurement of flowwas calculated for each breath. This signal, together with the pulmonarydriving pressure changes, which were collected using a Sensym pressuretransducer (#TRD4100), was connected via a Buxco (MAX 2270) preamplifierto a data collection interface (#'s SFT3400 and SFT3813). All otherpulmonary parameters were derived from these two inputs.

Baseline values were collected for 5 minutes, after which time theguinea pigs were challenged with ACh. ACh (0.1 mg/mL) was infusedintravenously for 1 minute from a syringe pump (sp210iw, World PrecisionInstruments, Inc., Sarasota, Fla.) at the following doses and prescribedtimes from the start of the experiment: 1.9 μg/minute at 5 minutes, 3.8μg/minute at 10 minutes, 7.5 μg/minute at 15 minutes, 15.0 μg/minute at20 minutes, 30 μg/minute at 25 minutes and 60 μg/minute at 30 minutes.If resistance or compliance had not returned to baseline values at 3minutes following each ACh dose, the guinea pig's lungs were inflated 3times with 4 mL of air from a 10 mL calibration syringe. Recordedpulmonary parameters included respiration frequency (breaths/minute),compliance (mL/cm H₂O) and pulmonary resistance (cm H₂O/mL per second).Once the pulmonary function measurements were completed at minute 35 ofthis protocol, the guinea pig was removed from the plethysmograph andeuthanized by carbon dioxide asphyxiation.

The data were evaluated in one or both of the following ways:

(a) Pulmonary resistance (R_(L), cm H₂O/mL per second) was calculatedfrom the ratio of “change in pressure” to “the change in flow.” TheR_(L) response to ACh (60 μg/min, IH) was computed for the vehicle andthe test compound groups. The mean ACh response in vehicle-treatedanimals, at each pre-treatment time, was calculated and used to compute% inhibition of ACh response, at the corresponding pre-treatment time,at each test compound dose. Inhibition dose-response curves for ‘R_(L)’were fitted with a four parameter logistic equation using GraphPadPrism, version 3.00 for Windows (GraphPad Software, San Diego, Calif.)to estimate bronchoprotective ID₅₀ (dose required to inhibit the ACh (60μg/min) bronchoconstrictor response by 50%). The equation used was asfollows:Y=Min+(Max−Min)/(1+10^(((log ID50-X)*Hillslope)))where X is the logarithm of dose, Y is the response (% Inhibition of AChinduced increase in R_(L)). Y starts at Min and approachesasymptotically to Max with a sigmoidal shape.

(b) The quantity PD₂, which is defined as the amount of ACh or histamineneeded to cause a doubling of the baseline pulmonary resistance, wascalculated using the pulmonary resistance values derived from the flowand the pressure over a range of ACh or histamine challenges using thefollowing equation (which is derived from a equation used to calculatePC₂₀ values described in American Thoracic Society. Guidelines formethacholine and exercise challenge testing-1999. Am J Respir Crit CareMed. 161: 309-329 (2000)):${PD}_{2} = {{antilog}\left\lbrack {{\log\quad C_{1}} + \frac{\left( {{\log\quad C_{2}} - {\log\quad C_{1}}} \right)\left( {{2R_{0}} - R_{1}} \right)}{R_{2} - R_{1}}} \right\rbrack}$where:

-   -   C₁=concentration of ACh or histamine preceding C₂    -   C₂=concentration of ACh or histamine resulting in at least a        2-fold increase in pulmonary resistance (R_(L))    -   R₀=Baseline R_(L) value    -   R₁=R_(L) value after C₁    -   R₂=R_(L) value after C₂

An efficacious dose was defined as a dose that limited thebronchrestriction response to a 50 μg/mL dose of ACh to a doubling ofthe baseline pulmonary resistance (PD₂₍₅₀₎).

Statistical analysis of the data was performed using a two-tailedStudents t-test. A P-value<0.05 was considered significant.

Generally, test compounds having a PD₂₍₅₀₎ less than about 200 μg/mL forACh-induced bronchoconstriction at 1.5 hours post-dose in this assay arepreferred. For example, the compound of Example 1 was found to have aPD₂₍₅₀₎ less than about 200 μg/mL for ACh-induced bronchoconstriction at1.5 hours post-dose.

Assay 4 Inhalation Guinea Pig Salivation Assay

Guinea pigs (Charles River, Wilmington, Mass.) weighing 200-350 g wereacclimated to the in-house guinea pig colony for at least 3 daysfollowing arrival. Test compound or vehicle were dosed via inhalation(IH) over a 10 minute time period in a pie shaped dosing chamber (R&SMolds, San Carlos, Calif.). Test solutions were dissolved in sterilewater and delivered using a nebulizer filled with 5.0 mL of dosingsolution. Guinea pigs were restrained in the inhalation chamber for 30minutes. During this time, guinea pigs were restricted to an area ofapproximately 110 sq. cm. This space was adequate for the animals toturn freely, reposition themselves, and allow for grooming. Following 20minutes of acclimation, guinea pigs were exposed to an aerosol generatedfrom a LS Star Nebulizer Set (Model 22F51, PARI Respiratory Equipment,Inc. Midlothian, Va.) driven by house air at a pressure of 22 psi. Uponcompletion of nebulization, guinea pigs were evaluated at 1.5, 6, 12,24, 48, or 72 hrs after treatment.

Guinea pigs were anesthetized one hour before testing with anintramuscular (IM) injection of a mixture of ketamine 43.75 mg/kg,xylazine 3.5 mg/kg, and acepromazine 1.05 mg/kg at an 0.88 mL/kg volume.Animals were placed ventral side up on a heated (37° C.) blanket at a 20degree incline with their head in a downward slope. A 4-ply 2×2 inchgauze pad (Nu-Gauze General-use sponges, Johnson and Johnson, Arlington,Tex.) was inserted in the guinea pig's mouth. Five minutes later, themuscarinic agonist pilocarpine (3.0 mg/kg, SC) was administered and thegauze pad was immediately discarded and replaced by a new pre-weighedgauze pad. Saliva was collected for 10 minutes, at which point the gauzepad was weighed and the difference in weight recorded to determine theamount of accumulated saliva (in mg). The mean amount of salivacollected for animals receiving the vehicle and each dose of testcompound was calculated. The vehicle group mean was considered to be100% salivation. Results were calculated using result means (n=3 orgreater). Confidence intervals (95%) were calculated for each dose ateach time point using two-way ANOVA. This model is a modified version ofthe procedure described in Rechter, “Estimation of anticholinergic drugeffects in mice by antagonism against pilocarpine-induced salivation”Ata Pharmacol Toxicol 24:243-254 (1996).

The mean weight of saliva in vehicle-treated animals, at eachpre-treatment time, was calculated and used to compute % inhibition ofsalivation, at the corresponding pre-treatment time, at each dose. Theinhibition dose-response data were fitted to a four parameter logisticequation using GraphPad Prism, version 3.00 for Windows (GraphPadSoftware, San Diego, Calif.) to estimate anti-sialagogue ID₅₀ (doserequired to inhibit 50% of pilocarpine-evoked salivation). The equationused was as follows:Y=Min+(Max-Min)/(1+10^(((log ID50-X)*Hillslope)))where X is the logarithm of dose, Y is the response (% inhibition ofsalivation). Y starts at Min and approaches asymptotically to Max with asigmoidal shape.

The ratio of the anti-sialagogue ID₅₀ to bronchoprotective ID₅₀ was usedto compute the apparent lung selectivity index of the test compound.Generally, compounds having an apparent lung selectivity index greaterthan about 5 are preferred. For example, in this assay, the compound ofExample 1 had an apparent lung-selectivity index greater than about 5.

Assay 5 Methacholine-Induced Depressor Responses in Conscious GuineaPigs

Healthy, adult, male Sprague-Dawley guinea pigs (Harlan, Indianapolis,Ind.), weighing between 200 and 300 g were used in these studies. Underisoflurane anesthesia (to effect), animals were instrumented with commoncarotid artery and jugular vein catheters (PE-50 tubing). The catheterswere exteriorized utilizing a subcutaneous tunnel to the subscapulararea. All surgical incisions were sutured with 4-0 Ethicon Silk and thecatheters locked with heparin (1000 units/mL). Each animal wasadministered saline (3 mL, SC) at the end of surgery as well asbuprenorphine (0.05 mg/kg, IM). Animals were allowed to recover on aheating pad before being returned to their holding rooms.

Approximately 18 to 20 hours following surgery, the animals were weighedand the carotid artery catheter on each animal was connected to atransducer for recording arterial pressure. Arterial pressure and heartrate was recorded using a Biopac MP-100 Acquisition System. Animals wereallowed to acclimate and stabilize for a period of 20 minutes.

Each animal was challenged with MCh (0.3 mg/kg, IV) administered throughthe jugular venous line and the cardiovascular response was monitoredfor 10 minutes. The animals were then placed into the whole body dosingchamber, which was connected to a nebulizer containing the test compoundor vehicle solution. The solution was nebulized for 10 minutes using agas mixture of breathable air and 5% carbon dioxide with a flow rate of3 liters/minute. The animals were then removed from the whole bodychamber and returned to their respective cages. At 1.5 and 24 hourspost-dosing, the animals were re-challenged with MCh (0.3 mg/kg, IV) andthe hemodynamic response was determined. Thereafter, the animals wereeuthanized with sodium pentobarbital (150 mg/kg, IV).

MCh produces a decrease in mean arterial pressure (MAP) and decrease inheart rate (bradycardia). The peak decrease, from baseline, in MAP(depressor responses) was measured for each MCh challenge (before andafter IH dosing). The bradycardic effects were not used for analysissince these responses were not robust and reproducible. The effects oftreatment on the MCh responses are expressed as % inhibition (mean±/−SEM) of the control depressor responses. Two-way ANOVA with theappropriate post-hoc test was used to test the effects of treatment andpre-treatment time. The depressor responses to MCh were relativelyunchanged at 1.5 and 24 hours after inhalation dosing with vehicle.

The ratio of the anti-depressor ID₅₀ to bronchoprotective ID₅₀ was usedto compute apparent lung-selectivity of the test compound. Generally,compounds having an apparent lung-selectivity index greater than 5 arepreferred. For example, in this assay, the compound of Example 1 had anapparent lung-selectivity index greater than 5.

While the present invention has been described with reference tospecific aspects or embodiments thereof, it will be understood by thoseof ordinary skilled in the art that various changes can be made orequivalents can be substituted without departing from the true spiritand scope of the invention. Additionally, to the extent permitted byapplicable patent statues and regulations, all publications, patents andpatent applications cited herein are hereby incorporated by reference intheir entirety to the same extent as if each document had beenindividually incorporated by reference herein.

1-14. (canceled)
 15. A pharmaceutical composition comprising apharmaceutically acceptable carrier and a therapeutically effectiveamount of a compound of claim 27 or
 28. 16. The pharmaceuticalcomposition of claim 15, wherein the composition further comprises atherapeutically effective amount of an agent selected from β₂ adrenergicreceptor agonists, steroidal anti-inflammatory agents,phosphodiesterase-4 inhibitors, and combinations thereof.
 17. Thepharmaceutical composition of claim 16, wherein the compositioncomprises a therapeutically effective amount of a β₂ adrenergic receptoragonist and a steroidal anti-inflammatory agent. 18-23. (canceled)
 24. Amethod for treating a pulmonary disorder, the method comprisingadministering to a patient a therapeutically effective amount of acompound of claim 27 or
 28. 25. A method of producing bronchodilation ina patient, the method comprising administering to a patient abronchodilation-producing amount of a compound of claim 27 or
 28. 26. Amethod of treating chronic obstructive pulmonary disease or asthma, themethod comprising administering to a patient a therapeutically effectiveamount of a compound of claim 27 or
 28. 27. A compound of formula:


28. A pharmaceutically acceptable salt of a compound of formula: